Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

Abstract

Introduction Cadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy. Materials and Methods Degenerated lumbar disks (grade 3, Schneiderman's classification) ( n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks ( n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG ( n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination. Results Human fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks. Conclusion Human fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Yang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121 Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256