Previous genotype:phenotype mapping of the mouse and primate dentition revealed the presence of pre- and post-canine modules in mice and anthropoid primates, as well as molar and premolar submodules in anthropoid primates. We estimated phenotypic correlation matrices for species that sample broadly across Mammalia to test the hypothesis that these modules exist across a broader range of taxa and thereby represent a conserved mammalian trait. We calculated phenotypic correlation matrices from linear dental measurements of 419 individual specimens representing 5 species from 4 mammalian orders: Artiodactyla (Odocoileus hemionus), Carnivora (Canis latrans and Ursus americanus), Didelphimorphia (Didelphis virginiana), and Primates (Colobus guereza). Our results based on hierarchical clustering indicate a generally higher correlation within incisors and among post-canine teeth. However, the post-canine phenotypic correlation matrices do not consistently exhibit the premolar and molar submodularity observed in anthropoid primates. Additionally, we find evidence of sex differences in the Odocoileus phenotypic correlation matrices: Males of this species exhibit overall higher inter-trait correlations compared to females. Our overall findings support the interpretation that incisors and post-canine dentition represent different phenotypic modules, and that this architecture may be a conserved trait for mammals.

Following corneal injury, coordinated cellular and protein interactions occur at the wound site to restore tissue homeostasis. Regulation of this response is required to prevent the development of chronic inflammation, abnormal neovascularization, and fibrosis. The chemokine CCL2 and its primary receptor CCR2 are key regulators of the inflammatory and neovascular responses to injury. In this study, we investigated the role of macrophage-associated matrix metalloproteinase 12 (MMP12) in the regulation of CCL2 and CCR2 after corneal wounding. Using two corneal injury models, we examined the temporal and spatial expression of CCL2 and CCR2 in Mmp12^-/- and wild-type (WT) mice. Our data showed that MMP12 downregulated CCL2 and CCR2 expression in a manner dependent on the timing and mechanism of injury. We also examined the effect of CCL2 on the injury response in Mmp12^-/- and WT corneas. We found that macrophage infiltration and neovascularization following CCL2 blockade was significantly reduced in Mmp12^-/- corneas as compared with WT corneas. These findings indicate that MMP12 inhibits corneal inflammation and neovascularization after injury through its regulation of CCL2.

Corneal epithelial defects are a common cause of ocular morbidity and can result in corneal scarring if they do not heal properly. Matrix metalloproteinases (MMPs) are extracellular matrix proteinases that regulate multiple aspects of corneal repair. We have previously shown that MMP12 has a protective effect on corneal fibrosis through its regulation of neutrophil and macrophage infiltration and angiogenesis in a chemical injury model involving full thickness damage to the cornea. However, the role of MMP12 in injuries limited to the corneal epithelium is relatively unknown. This study investigates the reparative effects of MMP12 following isolated corneal epithelial injury. Using a corneal epithelial debridement injury model performed on corneas of wild-type (WT) mice, we show that Mmp12 is expressed early following corneal epithelial injury with highest expression levels at 8 h after injury and lower expression levels at 4 and 8 days after injury. We investigated whether MMP12 has an effect on the rate of epithelial repair and cell migration using in vivo and in vitro scratch assays performed on WT and Mmp12^-/- mice. We found that loss of MMP12 results in a slower scratch wound repair rate both in vivo and in vitro. We also found that corneas of Mmp12^-/- mice have decreased neutrophil infiltration following injury. Loss of MMP12, however, does not affect cell proliferation in the center of the wounds. These data support a role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration.

Mononuclear phagocytes (MP) have central importance in innate immunity, inflammation, and fibrosis. Recruited MPs, such as macrophages, are plastic cells and can switch from an inflammatory to a restorative phenotype during the healing process. However, the role of the MPs in corneal wound healing is not completely understood. The purpose of this study is to characterize the kinetics of recruited MPs and evaluate the role of macrophage metalloelastase (MMP12) in the healing process, using an in vivo corneal chemical injury model. Unwounded and wounded corneas of wild-type (WT) and Mmp12^-/- mice were collected at 1, 3, and 6 days after chemical injury and processed for flow cytometry analysis. Corneal MP phenotype significantly changed over time with recruited Ly6C^high (proinflammatory) cells being most abundant at 1 day post-injury. Ly6C^int cells were highly expressed at 3 days post-injury and Ly6C^neg (patrolling) cells became the predominant cell type at 6 days post-injury. CD11c⁺ dendritic cells were abundant in corneas from Mmp12^-/- mice at 6 days post-injury. These findings show the temporal phenotypic plasticity of recruited MPs and provide valuable insight into the role of the MPs in the corneal repair response, which may help guide the future development of MP-targeted therapies.

Purpose

Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases involved in wound healing processes, including neovascularization and fibrosis. We assessed MMP protein expression levels in diseased corneas of patients requiring penetrating and deep anterior lamellar keratoplasty. The purpose of this study was to test the hypothesis that upregulation of MMPs in diseased corneas is positively associated with clinical levels of corneal neovascularization and fibrosis.

Methods

Protein expression levels of nine individual MMPs were quantified simultaneously in human corneal lysates by using the Bio-Plex Pro Human MMP 9-Plex Panel and the MAGPIX technology. Measurements of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, and MMP13 were performed on diseased specimens from 21 patients undergoing corneal transplantation (17 for penetrating keratoplasty and 4 for deep anterior lamellar keratoplasty) and 6 normal control corneas.

Results

Luminex-based expression analysis revealed a significant overexpression of four of the nine MMPs tested (MMP2, MMP8, MMP12, and MMP13) in patient samples compared to control. Significant overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 was observed in diseased corneas with neovascularization compared with diseased corneas without neovascularization. Overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 also corresponded with the levels of corneal fibrosis. Finally, reduced expression of MMP3 was detected in keratoconus patients.

Conclusions

Multiple MMPs are expressed in the corneas of patients with chronic disease requiring keratoplasty even when the pathologic process appears to be clinically inactive. In particular, the expression of several MMPs (MMP2, MMP8, MMP12, and MMP13) is positively associated with increased levels corneal fibrosis and neovascularization.

Transparency of the human cornea is necessary for vision. Fuchs Endothelial Corneal Dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium, and a leading indication for corneal transplantation in developed countries. While the early onset, and rarer, form of FECD has been linked to COL8A2 mutations, the more common, late onset form of FECD has genetic mutations linked to only a minority of cases. Epigenetic modifications that occur in FECD are unknown. Here, we report on and compare the DNA methylation landscape of normal human corneal endothelial (CE) tissue and CE from FECD patients using the Illumina Infinium HumanMethylation450 (HM450) DNA methylation array. We show that DNA methylation profiles are distinct between control and FECD samples. Differentially methylated probes (10,961) were identified in the FECD samples compared with the control samples, with the majority of probes being hypermethylated in the FECD samples. Genes containing differentially methylated sites were disproportionately annotated to ontological categories involving cytoskeletal organization, ion transport, hematopoetic cell differentiation, and cellular metabolism. Our results suggest that altered DNA methylation patterns may contribute to loss of corneal transparency in FECD through a global accumulation of sporadic DNA methylation changes in genes critical to basic CE biological processes.

The dentition is an extremely important organ in mammals with variation in timing and sequence of eruption, crown morphology, and tooth size enabling a range of behavioral, dietary, and functional adaptations across the class. Within this suite of variable mammalian dental phenotypes, relative sizes of teeth reflect variation in the underlying genetic and developmental mechanisms. Two ratios of postcanine tooth lengths capture the relative size of premolars to molars (premolar-molar module, PMM), and among the three molars (molar module component, MMC), and are known to be heritable, independent of body size, and to vary significantly across primates. Here, we explore how these dental traits vary across mammals more broadly, focusing on terrestrial taxa in the clade of Boreoeutheria (Euarchontoglires and Laurasiatheria). We measured the postcanine teeth of N = 1,523 boreoeutherian mammals spanning six orders, 14 families, 36 genera, and 49 species to test hypotheses about associations between dental proportions and phylogenetic relatedness, diet, and life history in mammals. Boreoeutherian postcanine dental proportions sampled in this study carry conserved phylogenetic signal and are not associated with variation in diet. The incorporation of paleontological data provides further evidence that dental proportions may be slower to change than is dietary specialization. These results have implications for our understanding of dental variation and dietary adaptation in mammals.