Pregnancy Associated Plasma Protein A (PappA) Knockout Mice: In Vivo Disc Study

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INTRODUCTIONPast research in disc biology has highlighted the beneficial effects of growth factors, such as BMPs and IGF‐1, in vitro. Based on this research, it was proposed that exogenous IGF‐1 be used as a therapy for intervertebral disc degeneration (IDD). In the aging research field, suppressing the IGF‐1‐mediated signaling pathway has consistently decreased the incidence of age‐related disorders and increased lifespan of mammals and rodents. One model using PappA knockout mice has shown decreasing IGF‐1 signaling leads to positive effects on aging and longevity. Active PappA protease cleaves IGF binding protein 4 (IGFBP4), releasing active IGF‐1 which can bind to its receptor and exert downstream signaling effects. When you genetically remove PappA, IGF‐1 stays bound to IGFBP4. Here, we examined the discs of aged PAPPA−/− mice versus wild type (WT) to determine whether there was an effect on age‐associated IDD.METHODSThis study was conducted according to IACUC approved protocols at University of Pittsburgh. Genetic deletion of PAPP‐A protease in mice with a mixed C57BL/6‐129SV/E background was achieved by deletion of both alleles of PAPP‐A. Spine segments were harvested from WT and PAPPA−/− mice at 23 months. Disc proteoglycan content was assessed by safranin‐O‐histology and DMMB assay was used to quantify sulfated glycosaminoglycans (GAG) in lumbar NP tissue. Western blotting was performed to measure the disc aggrecan proteolytic fragments, and the catabolic markers MMP‐3 and ADAMTS‐4. Levels of disc cellular senescence (loss of lamin B1expression) was assessed by immunohistochemistry. Student’s t‐test was used to test significance between groups (p<0.05). Average values (n=4 mice) were shown with SEM.RESULTSCompared to WT, PAPPA−/− mice had decreased proteoglycan content and two times less disc GAG content (Figure 1). PAPPA−/− mice also had significantly decreased aggrecan fragmentation and decreased protein levels of MMP‐3 and ADAMTS‐4 in their discs compared to WT mice (Figure 2). Additionally, greater level of disc lamin B1 was seen in PAPPA−/− compared to WT mice, suggesting reduced disc cellular senescence in PAPPA−/− mice.DISCUSSIONThe role of IGF‐1 in disc health and aging is currently unresolved. PAPPA−/− mice have reduced matrix content but the aggrecan matrix present after aging appears to be less fragmented based on our findings. Lowered disc fragmentation corresponded to a decrease in disc catabolic factors MMP‐3 and ADAMTS‐4 protein in PAPPA−/− mice. Lamin B1 staining suggests there are less disc senescent cells in old PAPPA−/− mice compared to normally aged mice. Further studies are needed to determine the mechanism for the decreased disc catabolism and more full length aggrecan in aged PAPPA−/− mice compared to aged WT mice. Additionally, further studies should confirm whether young PAPPA−/− mice also have decreased GAG content or this loss occurs during the aging process.SIGNIFICANCEThe results of this study suggest the net effect of IGF‐1 regarding both anabolic and catabolic effects must be examined to fully understand if IGF‐1 is beneficial for disc homeostasis during age‐associated IDD.Support or Funding InformationR01‐AG044376‐01 & Ferguson Foundation GrantQualitative and quantitative results show decreased disc proteoglycan content, including aggrecan, in aged PAPPA −/− mice.Figure 1Despite less proteoglycan content, aged PAPPA −/− mice had less fragmented aggrecan and lower levels of catabolic markers, MMP‐3 and ADAMTS‐4 compared to aged WT mice.Figure 2