Limited versus total epithelial debridement ocular surface injury: Live fluorescence imaging of hemangiogenesis and lymphangiogenesis in Prox1-GFP/Flk1::Myr-mCherry mice.

BACKGROUND: Immunohistochemical staining experiments have shown that both hemangiogenesis and lymphangiogenesis occur following severe corneal and conjunctival injury and that the neovascularization of the cornea often has severe visual consequences. To better understand how hemangiogenesis and lymphangiogenesis are induced by different degrees of ocular injury, we investigated patterns of injury-induced corneal neovascularization in live Prox1-GFP/Flk1::myr-mCherry mice, in which blood and lymphatic vessels can be imaged simultaneously in vivo. METHODS: The eyes of Prox1-GFP/Flk1::myr-mCherry mice were injured according to four models based on epithelial debridement of the: A) central cornea (a 1.5-mm-diameter circle of tissue over the corneal apex), B) total cornea, C) bulbar conjunctiva, and D) cornea+bulbar conjunctiva. Corneal blood and lymphatic vessels were imaged on days 0, 3, 7, and 10 post-injury, and the percentages of the cornea containing blood and lymphatic vessels were calculated. RESULTS: Neither central corneal nor bulbar conjunctival debridement resulted in significant vessel growth in the mouse cornea, whereas total corneal and corneal+bulbar conjunctival debridement did. On day 10 in the central cornea, total cornea, bulbar conjunctiva, and corneal+bulbar conjunctival epithelial debridement models, the percentage of the corneal surface that was occupied by blood vessels (hemangiogenesis) was 1.9±0.8%, 7.14±2.4%, 2.29±1%, and 15.05±2.14%, respectively, and the percentage of the corneal surface that was occupied by lymphatic vessels (lymphangiogenesis) was 2.45±1.51%, 4.85±0.95%, 2.95±1.27%, and 4.15±3.85%, respectively. CONCLUSIONS: Substantial corneal debridement was required to induce corneal neovascularization in the mouse cornea, and the corneal epithelium may therefore be partially responsible for maintaining corneal avascularity. GENERAL SIGNIFICANCE: Our study demonstrates that GFP/Flk1::myr-mCherry mice are a useful model for studying coordinated hemangiogenic and lymphangiogenic responses.

MMP14 Cleavage of VEGFR1 in the Cornea Leads to a VEGF-Trap Antiangiogenic Effect.

PURPOSE: To determine the possible antiangiogenic effect of metalloproteinase (MMP) 14 cleavage of vascular endothelial growth factor receptor 1 (VEGFR1) in the cornea. METHODS: Recombinant mouse (rm) VEGFR1 was incubated with various concentrations of recombinant MMP14 to examine proteolysis in vitro. The reaction mixture was analyzed by SDS-PAGE and stained with Coomassie blue. The fragments resulting from rmVEGFR1 cleavage by MMP14 were subjected to Edman degradation, and the amino acid sequences were aligned with rmVEGFR1 sequences. Surface plasmon resonance was used to determine the equilibrium dissociation constant (KD) between MMP14 and rmVEGFR1. The KD value of rmVEGFR1 and the 59.8-kDa cleavage product binding to VEGF-A165 was also determined. Cell proliferation assays were performed in the presence of VEGF-A165 plus the 59.8-kDa VEGFR1 fragment or VEGF-A165 alone. RESULTS: Matrix metalloproteinase 14 binds and cleaves rmVEGFR1 to produce 59.8-kDa (N-terminal fragment, Ig domains 1-5), 35-kDa (C-terminal fragment containing IgG and His-tag), and 21-kDa (Ig domains 6-7) fragments. The 59.8-kDa fragment showed binding to VEGF-A165 and inhibited VEGF-induced endothelial cell mitogenesis. CONCLUSIONS: Our findings suggest that VEGFR1 cleavage by MMP14 in the cornea leads to a VEGF-trap effect, reducing the proangiogenic effect of VEGF-A165, thereby reducing corneal angiogenesis.

Levels of homology and the problem of neocortex.

The neocortex is found only in mammals, and the fossil record is silent on how this soft tissue evolved. Understanding neocortex evolution thus devolves to a search for candidate homologous neocortex traits in the extant nonmammalian amniotes. The difficulty is that homology is based on similarity, and the six-layered neocortex structure could hardly be more dissimilar in appearance from the nuclear organization that is so conspicuous in the dorsal telencephalon of birds and other reptiles. Recent molecular data have, however, provided new support for one prominent hypothesis, based on neuronal circuits, that proposes the principal neocortical input and output cell types are a conserved feature of amniote dorsal telencephalon. Many puzzles remain, the greatest being understanding the selective pressures and molecular mechanisms that underlie such tremendous morphological variation in telencephalon structure.

Simultaneous in vivo imaging of blood and lymphatic vessel growth in Prox1-GFP/Flk1::myr-mCherry mice.

The ability to visually observe angiogenesis and lymphangiogenesis simultaneously and repeatedly in living animals would greatly enhance our understanding of the inter-dependence of these processes. To generate a mouse model that allows such visualization via in vivo fluorescence imaging, we crossed Prox1-GFP mice with Flk1::myr-mCherry mice to generate Prox1-GFP/Flk1::myr-mCherry mice, in which lymphatic vessels emit green fluorescence and blood vessels emit red fluorescence. Corneal neovascularization was induced in these mice using three injury models: implantation of a vascular endothelial growth factor (VEGF) pellet, implantation of a basic fibroblast growth factor (bFGF) pellet, and alkali burn injury. Vessel growth was observed in vivo by stereomicroscopy on days 0, 3, 7 and 10 after pellet implantation or alkali injury as well as in flat-mounted corneas via confocal microscopy after the final in vivo imaging time point. We observed blood and lymphatic vessel growth in all three models, with the most significant growth occurring from days 0-7. Upon VEGF stimulation, the growth kinetics of blood and lymphatic vessels were similar. Blood vessels exhibited similar growth patterns in VEGF- and bFGF-stimulated corneas. Alkali burn injury induced robust angiogenesis and lymphangiogenesis. The intrinsic fluorescence of blood and lymphatic endothelial cells in Prox1-GFP/Flk1::myr-mCherry mice permitted simultaneous in vivo imaging of angiogenesis and lymphangiogenesis. This allowed us to differentiate the processes as well as observe their inter-dependence, and will be valuable in development of therapies targeting angiogenesis and/or lymphangiogenesis.

Evidence for the Involvement of MMP14 in MMP2 Processing and Recruitment in Exosomes of Corneal Fibroblasts.

PURPOSE: Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechanisms are poorly understood. In this study, we investigated exosomal transport of MMP14 and its target, MMP2, from corneal fibroblasts to vascular endothelial cells as a possible mechanism governing MMP14 activity in corneal angiogenesis. METHODS: We isolated MMP14-containing exosomes from corneal fibroblasts by sucrose density gradient and evaluated exosome content and purity by Western blot analysis. We then investigated exosome transport in vitro from corneal fibroblasts to two populations of vascular endothelial cells, human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAECs). Western blot analysis and gelatin zymography were used to determine levels of MMP14 and MMP2, respectively, in exosomal fractions derived from cultured wild-type, MMP14 enzymatic domain-deficient (MMP14Δexon4), and MMP14-null corneal fibroblasts. RESULTS: Matrix metalloproteinase 14-containing exosomes isolated from corneal fibroblasts were readily taken up in vitro by HUVECs and CPAECs. We found that MMP14 was enriched in exosomal fractions of cultured corneal fibroblasts. Moreover, loss of the MMP14 enzymatic domain resulted in accumulation of pro-MMP2 protein in exosomes, whereas MMP2 was nearly undetectable in exosomes of MMP14-null fibroblasts. CONCLUSIONS: Our results indicate that exosomes secreted by corneal fibroblasts can transport proteins, including MMP14, to vascular endothelial cells. In addition, recruitment of MMP2 into corneal fibroblast exosomes is an active process that depends, at least in part, on the presence of MMP14. The role of exosomal MMP14 transport in corneal angiogenesis has important implications for therapeutic applications targeting angiogenic processes in the cornea.

Involvement of lysosomal degradation in VEGF-C-induced down-regulation of VEGFR-3.

The vascular endothelial growth factor (VEGF)-C-induced down-regulation of VEGF receptor (VEGFR)-3 is important in lymphangiogenesis. Here, we demonstrate that VEGF-C, -D, and -C156S, but not VEGF-A, down-regulate VEGFR-3. VEGF-C stimulates VEGFR-3 tyrosyl phosphorylation and transient phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinases in lymphatic endothelial cells. VEGF-C-induced down-regulation of VEGFR-3 was blocked by a VEGF-C trap, tyrosine kinase inhibitor, and leupeptin, pepstatin, and E64 (LPE), but was unaffected by Notch 1 activator and γ-secretase inhibitors. Our findings indicate that VEGF-C down-regulates VEGFR-3 in lymphatic endothelial cells through VEGFR-3 kinase activation and, in part, via lysosomal degradation.

Cell-type homologies and the origins of the neocortex.

The six-layered neocortex is a uniquely mammalian structure with evolutionary origins that remain in dispute. One long-standing hypothesis, based on similarities in neuronal connectivity, proposes that homologs of the layer 4 input and layer 5 output neurons of neocortex are present in the avian forebrain, where they contribute to specific nuclei rather than to layers. We devised a molecular test of this hypothesis based on layer-specific gene expression that is shared across rodent and carnivore neocortex. Our findings establish that the layer 4 input and the layer 5 output cell types are conserved across the amniotes, but are organized into very different architectures, forming nuclei in birds, cortical areas in reptiles, and cortical layers in mammals.

Molecular analysis of neocortical layer structure in the ferret.

Molecular markers that distinguish specific layers of rodent neocortex are increasingly employed to study cortical development and the physiology of cortical circuits. The extent to which these markers represent general features of neocortical cell type identity across mammals, however, is unknown. To assess the conservation of layer markers more broadly, we isolated orthologs for 15 layer-enriched genes in the ferret, a carnivore with a large, gyrencephalic brain, and analyzed their patterns of neocortical gene expression. Our major findings are: 1) Many but not all layer markers tested show similar patterns of layer-specific gene expression between mouse and ferret cortex, supporting the view that layer-specific cell type identity is conserved at a molecular level across mammalian superorders; 2) Our panel of deep layer markers (ER81/ETV1, SULF2, PCP4, FEZF2/ZNF312, CACNA1H, KCNN2/SK2, SYT6, FOXP2, CTGF) provides molecular evidence that the specific stratifications of layers 5 and 6 into 5a, 5b, 6a, and 6b are also conserved between rodents and carnivores; 3) Variations in layer-specific gene expression are more pronounced across areas of ferret cortex than between homologous areas of mouse and ferret cortex; 4) This variation of area gene expression was clearest with the superficial layer markers studied (SERPINE2, MDGA1, CUX1, UNC5D, RORB/NR1F2, EAG2/KCNH5). Most dramatically, the layer 4 markers RORB and EAG2 disclosed a molecular sublamination to ferret visual cortex and demonstrated a molecular dissociation among the so-called agranular areas of the neocortex. Our findings establish molecular markers as a powerful complement to cytoarchitecture for neocortical layer and cell-type comparisons across mammals.

Avian brains and a new understanding of vertebrate brain evolution.

We believe that names have a powerful influence on the experiments we do and the way in which we think. For this reason, and in the light of new evidence about the function and evolution of the vertebrate brain, an international consortium of neuroscientists has reconsidered the traditional, 100-year-old terminology that is used to describe the avian cerebrum. Our current understanding of the avian brain - in particular the neocortex-like cognitive functions of the avian pallium - requires a new terminology that better reflects these functions and the homologies between avian and mammalian brains.

Revised nomenclature for avian telencephalon and some related brainstem nuclei.

The standard nomenclature that has been used for many telencephalic and related brainstem structures in birds is based on flawed assumptions of homology to mammals. In particular, the outdated terminology implies that most of the avian telencephalon is a hypertrophied basal ganglia, when it is now clear that most of the avian telencephalon is neurochemically, hodologically, and functionally comparable to the mammalian neocortex, claustrum, and pallial amygdala (all of which derive from the pallial sector of the developing telencephalon). Recognizing that this promotes misunderstanding of the functional organization of avian brains and their evolutionary relationship to mammalian brains, avian brain specialists began discussions to rectify this problem, culminating in the Avian Brain Nomenclature Forum held at Duke University in July 2002, which approved a new terminology for avian telencephalon and some allied brainstem cell groups. Details of this new terminology are presented here, as is a rationale for each name change and evidence for any homologies implied by the new names. Revisions for the brainstem focused on vocal control, catecholaminergic, cholinergic, and basal ganglia-related nuclei. For example, the Forum recognized that the hypoglossal nucleus had been incorrectly identified as the nucleus intermedius in the Karten and Hodos (1967) pigeon brain atlas, and what was identified as the hypoglossal nucleus in that atlas should instead be called the supraspinal nucleus. The locus ceruleus of this and other avian atlases was noted to consist of a caudal noradrenergic part homologous to the mammalian locus coeruleus and a rostral region corresponding to the mammalian A8 dopaminergic cell group. The midbrain dopaminergic cell group in birds known as the nucleus tegmenti pedunculopontinus pars compacta was recognized as homologous to the mammalian substantia nigra pars compacta and was renamed accordingly; a group of gamma-aminobutyric acid (GABA)ergic neurons at the lateral edge of this region was identified as homologous to the mammalian substantia nigra pars reticulata and was also renamed accordingly. A field of cholinergic neurons in the rostral avian hindbrain was named the nucleus pedunculopontinus tegmenti, whereas the anterior nucleus of the ansa lenticularis in the avian diencephalon was renamed the subthalamic nucleus, both for their evident mammalian homologues. For the basal (i.e., subpallial) telencephalon, the actual parts of the basal ganglia were given names reflecting their now evident homologues. For example, the lobus parolfactorius and paleostriatum augmentatum were acknowledged to make up the dorsal subdivision of the striatal part of the basal ganglia and were renamed as the medial and lateral striatum. The paleostriatum primitivum was recognized as homologous to the mammalian globus pallidus and renamed as such. Additionally, the rostroventral part of what was called the lobus parolfactorius was acknowledged as comparable to the mammalian nucleus accumbens, which, together with the olfactory tubercle, was noted to be part of the ventral striatum in birds. A ventral pallidum, a basal cholinergic cell group, and medial and lateral bed nuclei of the stria terminalis were also recognized. The dorsal (i.e., pallial) telencephalic regions that had been erroneously named to reflect presumed homology to striatal parts of mammalian basal ganglia were renamed as part of the pallium, using prefixes that retain most established abbreviations, to maintain continuity with the outdated nomenclature. We concluded, however, that one-to-one (i.e., discrete) homologies with mammals are still uncertain for most of the telencephalic pallium in birds and thus the new pallial terminology is largely devoid of assumptions of one-to-one homologies with mammals. The sectors of the hyperstriatum composing the Wulst (i.e., the hyperstriatum accessorium intermedium, and dorsale), the hyperstriatum ventrale, the neostriatum, and the archistriatum have been renamed (respectively) the hyperpallium (hypertrophied pallium), the mesopallium (middle pallium), the nidopallium (nest pallium), and the arcopallium (arched pallium). The posterior part of the archistriatum has been renamed the posterior pallial amygdala, the nucleus taeniae recognized as part of the avian amygdala, and a region inferior to the posterior paleostriatum primitivum included as a subpallial part of the avian amygdala. The names of some of the laminae and fiber tracts were also changed to reflect current understanding of the location of pallial and subpallial sectors of the avian telencephalon. Notably, the lamina medularis dorsalis has been renamed the pallial-subpallial lamina. We urge all to use this new terminology, because we believe it will promote better communication among neuroscientists. Further information is available at http://avianbrain.org

The Avian Brain Nomenclature Forum: Terminology for a New Century in Comparative Neuroanatomy.

Many of the assumptions of homology on which the standard nomenclature for the cell groups and fiber tracts of avian brains have been based are in error, and as a result that terminology promotes misunderstanding of the functional organization of avian brains and their evolutionary relationship to mammalian brains. Recognizing this problem, a number of avian brain researchers began an effort to revise the terminology, which culminated in the Avian Brain Nomenclature Forum, held at Duke University from July 18 to 20, 2002. In the new terminology approved at this Forum, the flawed conception that the telencephalon of birds consists nearly entirely of a hypertrophied basal ganglia has been purged from the telencephalic terminology, and the actual parts of the basal ganglia and its brainstem afferent cell groups have been given names reflecting their now evident homologies. The telencephalic regions that were erroneously named to reflect presumed homology to mammalian basal ganglia were renamed as parts of the pallium, using prefixes that retained most established abbreviations (to maintain continuity with the replaced nomenclature). Details of this meeting and its major conclusions are presented in this paper, and the details of the new terminology and its basis are presented in a longer companion paper. We urge all to use this new terminology, because we believe it will promote better communication among neuroscientists.