Comprehensive Genome-Wide Transcriptomic Analysis of Immature Articular Cartilage following Ischemic Osteonecrosis of the Femoral Head in Piglets

<div><p>Objective</p><p>Ischemic osteonecrosis of the femoral head (ONFH) in piglets results in an ischemic injury to the immature articular cartilage. The molecular changes in the articular cartilage in response to ONFH have not been investigated using a transcriptomic approach. The purpose of this study was to perform a genome-wide transcriptomic analysis to identify genes that are upregulated in the immature articular cartilage following ONFH.</p><p>Methods</p><p>ONFH was induced in the right femoral head of 6-week old piglets. The unoperated femoral head was used as the normal control. At 24 hours (acute ischemic-hypoxic injury), 2 weeks (avascular necrosis in the femoral head) and 4 weeks (early repair) after surgery (n = 4 piglets/time point), RNA was isolated from the articular cartilage of the femoral head. A microarray analysis was performed using Affymetrix Porcine GeneChip Array. An enrichment analysis and functional clustering of the genes upregulated due to ONFH were performed using DAVID and STRING software, respectively. The increased expression of selected genes was confirmed by a real-time qRTPCR analysis.</p><p>Results</p><p>Induction of ONFH resulted in the upregulation of 383 genes at 24 hours, 122 genes at 2 weeks and 124 genes at 4 weeks compared to the normal controls. At 24 hours, the genes involved in oxidoreductive, cell-survival, and angiogenic responses were significantly enriched among the upregulated genes. These genes were involved in HIF-1, PI3K-Akt, and MAPK signaling pathways. At 2 weeks, secretory and signaling proteins involved in angiogenic and inflammatory responses, PI3K-Akt and matrix-remodeling pathways were significantly enriched. At 4 weeks, genes that represent inflammatory cytokines and chemokine signaling pathways were significantly enriched. Several index genes (genes that are upregulated at more than one time point following ONFH and are known to be important in various biological processes) including <i>HIF-1A</i>, <i>VEGFA</i>, <i>IL-6</i>, <i>IL6R</i>, <i>IL-8</i>, <i>CCL2</i>, <i>FGF2</i>, <i>TGFB2</i>, <i>MMP1</i>, <i>MMP3</i>, <i>ITGA5</i>, <i>FN and Col6A1</i> were upregulated in the immature articular cartilage following ONFH. A qRTPCR analysis of selected genes confirmed the upregulated expression observed in the microarray analysis.</p><p>Conclusion</p><p>Immature articular cartilage responds to ONFH by the upregulation of genes involved in hypoxic stress response, angiogenesis, matrix remodeling and inflammation. This study provides novel insights into the multi-faceted role of immature articular cartilage, with inflammation as a key component, following ONFH in piglets.</p></div

Response to hypoxic stress (HIF-1 pathway) and cell survival signaling pathways (PI3K-Akt, MAPK pathways) are the most significantly enriched functions in the immature articular cartilage at 24 hours following the induction of ONFH.

<p>The genes and the specific pathways that were significantly enriched (p<0.05, based on t-tests, Bonferroni, FDR) are listed. The network diagram (STRING10 software) indicates a close relationship of different genes based on the functional roles in biological pathways. The clusters of genes indicating specific biological functions (cell survival and signaling, matrix and growth factors, hypoxic stress response) are indicated using boxes with dashed lines. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines indicate closer relationship. This evidence plot utilizes multiple lines to indicate stronger evidence for the proximity in a functional relationship.</p

Angiogenesis, matrix remodeling and inflammation are the most significantly enriched functions in the immature articular cartilage at 2 weeks following the induction of ONFH.

<p>The genes and the specific pathways that were significantly enriched (p<0.05, based on t-tests, Bonferroni, FDR) are listed. The STRING10 network diagram was used to indicate the closeness of relationship of different genes based on the functional roles in biological pathways. The clusters of genes indicating matrix-related functions (e.g. <i>FN</i>, <i>TNC</i>, <i>MMP1</i>, <i>MMP3</i>), angiogenesis (e.g. <i>VEGF</i>) and inflammation (e.g. <i>IL8</i>, <i>OSMR</i>) are shown. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines indicate closer relationship. The evidence plot utilizes multiple lines to indicate stronger evidence for the proximity in a functional relationship.</p

Index genes representing the major roles of the immature articular cartilage.

<p>The STRING10 network diagram indicates the functional clustering of the index genes. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines connect the closely related genes. Multiple connectors indicate stronger evidence for the functional relationship.</p

Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 2 weeks following ONFH.

<p>Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 2 weeks following ONFH.</p

Selected index genes showed increased expression in the immature articular cartilage by qRTPCR analysis.

<p>The microarray results were validated by real-time qRTPCR analysis of the expression of selected index genes at 24 hours, 2 weeks and 4 weeks following the induction of ONFH. <b>(A)</b> The hypoxic response (<i>HIFA</i>, hypoxia inducible factor 1 alpha), angiogenesis (<i>VEGFA</i>, vascular endothelial growth factor alpha), <b>(B)</b> cytokines/cytokine receptors (<i>IL6/IL6R</i>, interleukin 6/interleukin 6 receptor), <b>(C)</b> chemokines (<i>IL8</i>, interleukin 8, CCL2, chemokine (C-C) motif ligand-2 <b>(D)</b> matrix related factors (<i>FN</i>, fibronectin; <i>ITGA5</i>, integrin alpha v subunit), <b>(E)</b> growth factors (<i>FGF2</i>, fibroblast growth factor-2) and <b>(F)</b> transcription factor (<i>NFkB p65</i>, nuclear factor kappa b, subunit p65) were confirmed by qRTPCR. The mRNA levels were normalized to 18sRNA and the data is shown as fold difference relative to normal articular cartilage. A total of n = 4 pigs were used per time point. Statistical analysis was performed using ANOVA and Tukey’s multiple comparison tests. P value * <0.05, **<0.01, ***<0.001, ****<0.0001</p

Genes involved in inflammatory response are significantly enriched in the immature articular cartilage at 4 weeks following the induction of ONFH.

<p>The genes and the pathways that were significantly enriched (p<0.05, based on t-tests, Bonferroni, FDR) belong to inflammation. The STRING10 network diagram indicates that the different genes are closely related to the inflammatory responses. The clusters of genes indicating chemokines/cytokines/receptors and the relationship to growth factors and matrix related genes show the functional link between matrix and recruitment of inflammatory cells. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines indicate closer relationship. The evidence plot utilizes multiple lines to indicate stronger evidence for the proximity in a functional relationship.</p

Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 24 hours following ONFH.

<p>Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 24 hours following ONFH.</p

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

<div><p>Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.</p></div

Lack of Cbl-PI3K interaction results in increased osteoblast surface, incorporation of dynamic bone labels, and up-regulation of master transcription factor Osterix in remodeling fracture calluses.

<p>Mid-saggital sections of fractured femora were stained with Safranin O and Fast Green, and counterstained with Hematoxylin. <b>A.</b> Representative sections at 7, 14, 21, and 28 days post-fracture showing bony regions of fracture calluses. <b>B.</b> Osteoblast surface over bone surface (ObS/BS) measured by static histomorphometry. n = 6, *p<0.05 vs. WT. <b>C.</b> Mice were injected with Alizarin Complexone (red) 4 days prior to sacrifice, and Calcein (green) 1 day prior to sacrifice. Mid-sagittal sections of fractured femora were used to image incorporation of dynamic bone labels in the center of the fracture calluses at 14 and 21 days post-fracture. Cells from sections of calluses at 7, 14, and 21 days post-fracture were harvested by LCM, and expression of <b>D.</b> Runx2, and <b>E.</b> Osterix were quantified by qRT-PCR n = 6, *p<0.05 vs. WT.</p