Vascular Endothelial Growth Factor Expression in the Murine Dental Pulp During Aging and Dentin Regeneration
Parent, Sara N.
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Vascular endothelial growth factor (VEGF) is an important pro-angiogenic growth factor that is essential for maintenance of vascular networks throughout the body, including those in the dental pulp. In addition, it has been suggested that VEGF may play a role in dentin regeneration and mineralization. As well studied as VEGF is, currently there are no studies looking at the effects of aging on VEGF expression in the dental pulp. We proposed a study of the expression levels of VEGF in the dental pulp during aging using a mouse model to test whether VEGF expression decreases with age and whether this decrease causes a decrease in pulpal vascular density and tertiary dentin formation. Tertiary dentin is formed as an internal reparative response to injury to the tooth, which can involve stem cell recruitment from the dental pulp. However, better understanding of this process is needed to develop stem cell and tissue regeneration applications to improve the organization and formation of tertiary dentin following injury. Mandibles from mice in six separate age groups were collected and histological analysis was performed. Mandibular molar sections were stained for antibodies for VEGFR-2, VEGFR-3, VEGF-A, VEGF-C, LYVE-1 (a lymphatic marker) and BS-1 (a vascular marker). Mandibular molars were also isolated from three separate age groups and collected for gene expression analysis using Q-RT-PCR. The immunofluorescence results from the vascularity study showed a significant decrease in blood vessel density with the highest levels found in the neonate and the lowest found in the most aged samples. There was also a distinct qualitative difference in the expression of VEGF-A in the dental pulp and odontoblast layers of samples from each age group that followed the same trend as the vasculature. Lymphatics were not detected in any of the age groups. Q-RT-PCR results showed that there was a decrease in gene expression in vWF (a blood vessel marker), VE-cadherin (a blood vessel marker), VEGFR-3, VEGF-A and VEGF-C from the neonatal to senescent age groups. VEGFR-1 and -2 were only detected at low levels in the neonatal age group. Surprisingly VEGF-C and VEGFR-3 were highly expressed in the dental pulp and although their expressions also decrease with aging, they were the only VEGF ligand and receptor detectable in the uninjured adult mouse molar. For the second part of the study, to determine if VEGF played a role in dentin regeneration, the first maxillary molars of nine mice were drilled to induce a wound healing response in the tooth. They were allowed time to heal and then the maxillae were collected and stained with 3,3'-diaminobenzidine for VEGF-A and VEGFR-2 expression at three time points post-injury. The histological sections from injured molars showed a dramatic increase in VEGF-A expression fifteen minutes after injury that dissipated with time and returned to near normal levels after four weeks post-injury. VEGFR-2 expression was also elevated soon after injury and then gradually returned to pre-injury levels. Current findings of this study suggest that although the expression of VEGF-A and its receptor VEGFR-2, which has been traditionally viewed as the most potent pro- angiogenic signaling pathway, does decrease with age, this ligand and receptor may play a more important role in regulating the response to tooth injury and dental regeneration. In contrast, VEGFR-3 and its ligand VEGF-C, which have traditionally been associated with only lymphangiogenesis, may be contributing to the regulation of angiogenesis in the dental pulp.
- Dentistry