Susan H. Kidson

Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells.

Hypericin, the major component of St. Johns Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.

Melanomas display increased cytoprotection to hypericin-mediated cytotoxicity through the induction of autophagy

Photodynamic therapy (PDT) as a regime for melanoma is of limited success due to factors such as the efficacy of the photosensitizer used, penetration depth and the presence of pigment. We characterised a pigmented and an unpigmented melanoma cell line with respect to their phenotypes. Cell viability was assessed after exposure to hypericin, a UVA‐activated photosensitizer. Exposure to 3 μM activated hypericin induced a cytoprotective (autophagic) response from both cell lines. However, the pigmented cells accumulated a large amount of glycogen in their cytoplasm. We hypothesise that the treatment induces an initial cytoprotective response through autophagy, but with increased stress results in a different mode of cell death in pigmented melanoma cells from unpigmented cells. These results indicate that hypericin‐PDT could be an adjuvant therapy for melanoma.

Structure of the integument of southern right whales, Eubalaena australis

Skin (integument) anatomy reflects adaptations to particular environments. It is hypothesized that cetacean (whale) integument will show unique anatomical adaptations to an aquatic environment, particularly regarding differences in temperature, density, and pressure. In this study, the gross and histological structure of the southern right whale integument is described and compared with terrestrial mammals and previous descriptions of mysticete (baleen whale) and odontocete (toothed whale) species. Samples were taken of the integument of 98 free‐swimming southern right whales, Eubalaena australis, and examined by both light and electron microscopy. Results show that three epidermal layers are present, with the stratum corneum being parakeratotic in nature. As in bowhead whales, southern right whales possess an acanthotic epidermis and a notably thick hypodermis, with epidermal rods and extensive papillomatosis. However, unlike bowhead whales, southern right whales possess an uninterrupted hypodermal layer. Surprisingly, the integument of balaenids (right and bowhead mysticetes) in general is more like that of odontocetes than that of the more closely related balaenopterids (rorqual mysticetes). Similarities to odontocetes were found specifically in the collagen fibers in a fat‐free zone of the reticular dermal layer and the elastic fibers in the dermal and hypodermal layers. Callosities, a distinctive feature of this genus, have a slightly thicker stratum corneum and are usually associated with hairs that have innervated and vascularized follicles. These hairs may function as vibrissae, thus aiding in aquatic foraging by allowing rapid detection of changes in prey density. Although the thick insulatory integument makes right whales bulky and slow‐moving, it is an adaptation for living in cold water. Epidermal thickness, presence of epidermal rods, and callosities may act as barriers against mechanical injury from bodily contact with conspecifics or hard surfaces in the environment (e.g., rocks, ice). Anat Rec, 290:596–613, 2007.

Proliferation and cell shape changes during ciliary body morphogenesis in the mouse

Very little is known about the structure and development of the ciliary processes in the mouse eye. Our scanning electron microscope (SEM) studies reveal that, unlike other mammals, the ciliary processes form an irregular pattern, crossing over and interweaving rather than lying parallel to one another. Histological and SEM studies from embryonic day (E) 14.5 to postnatal day (P) 7 reveal that the first morphological sign of the ciliary zone is an annular bulge; this is then gradually molded to form discrete ciliary processes. The striking similarity between the developing capillary network and the adult ciliary folds suggests that the patterning template for the ciliary processes could be the underlying capillary network. Cell proliferation measurements and cell height assessments indicated that one of the first events occurring during the morphogenesis of ciliary processes is a proliferative surge around P0 in the outer ciliary epithelium. It is likely that this surge together with increasing cell heights leads to a bulging of this layer. After a slight delay, the inner ciliary epithelium responds by proliferating and extending inward toward the lens. Final shaping of the ciliary processes is achieved through cell height reductions in the inner ciliary epithelium. Thus, in the mouse, the temporal correlation between mitotic and cell height changes during ciliary body morphogenesis suggests that these processes play an integral role in the shaping of ciliary processes. Developmental Dynamics 233:213–223, 2005.

Advances in imaging the blood and aqueous vessels of the ocular limbus

The vessels of the limbus play a pivotal role in the drainage of the major portion of aqueous humour from the anterior chamber. Aberrations in the limbal architecture can lead to raised intraocular pressure, which in turn can lead to blinding conditions such as glaucoma. Imaging these vessels in the normal eye, in development, and in conditions where there is anterior segment dysgenesis remains a challenge. Here we review the progress in limbal vessel imaging in the past 50 years and provide key information on their strengths and limitations. Included is an analysis of serial histological sectioning, ultrathin sections, microvascular perfusion with plastics and corrosion casting, X-ray microcomputed tomography, in vivo imaging including analysis of transgenic mice expressing GFP-vascular endothelium fusion proteins, in vivo microscopy imaging using fluorescent-labelled antibodies, slit-lamp microscopy and gonioscopy, fluorescein angiography, optical coherence tomography, and various labelling procedures for the vascular endothelium and the various forms of microscopy used to view these.

The role of the forkhead transcription factor, Foxc1, in the development of the mouse lacrimal gland.

The lacrimal gland produces secretions that lubricate and protect the cornea of the eye. Foxc1 encodes a forkhead/winged helix transcription factor required for the development of many embryonic organs. Autosomal dominant mutations in human FOXC1 cause eye disorders such as Axenfeld‐Rieger Syndrome and glaucoma iris hypoplasia, resulting from malformation of the anterior segment of the eye. We show here that lacrimal gland development is severely impaired in homozygous null Foxc1 mouse mutants, with reduced outgrowth and branching. Foxc1 is expressed in both the epithelium of the lacrimal gland and the surrounding mesenchyme. FGF10 stimulates the growth and branching morphogenesis in cultures of wild type and Foxc1 mutant gland epithelial buds. However, using micromass culture of lacrimal gland mesenchyme, we show that Bmp7 induces wild type mesenchyme cells to aggregate, but Foxc1 mutant cells do not respond. This study demonstrates that Foxc1 mediates the BMP signaling required for lacrimal gland development. Developmental Dynamics 235:1074–1080, 2006.

Terminal migration and early differentiation of melanocytes in embryonic chick skin.

The microenvironment is thought to play a key role in the control of neural crest cell diversification. To investigate its role in melanocyte differentiation we mapped the temporal and spatial distribution of pigmented melanocytes in embryonic chick skin and determined, by experimental means, the route taken by migrating melanocytes in the skin. We show that the New Hampshire Red/Black Australorp crossbreed exhibits melanization from 5 days of incubation (2 1/2 days earlier than is reported in other breeds). Contrary to previous reports our findings show that melanization is at first predominantly dermal. Both dermal and epidermal melanocyte numbers increase until Day 8, whereafter there is a dramatic decline in dermal melanocytes and by Day 10, melanocytes are almost exclusively located in the epidermis. Using homeotypic and heterotypic combinations of white and red/black dermis and epidermis we have demonstrated that premelanocytes arrive in the dermis of the trunk by Day 3 and begin to move into the epidermis from Day 4 onward. Results from these grafts and from tritium labeling studies strongly suggest that there is little or no reverse migration of premelanocytes from epidermis to dermis. Our findings indicate that overt melanocyte differentiation is not dependent on location in an epidermal environment, and that melanogenesis does not signify the end-stage in the migration process. Further, they suggest that the early dermal mesenchyme plays a key role in controlling melanogenesis.

Identification of Tgfβ1i4 as a downstream target of Foxc1

Craniofacial development is severely affected by null mutations in Foxc1, indicating a multifunctional role for Foxc1 in ocular, maxilla and mandible, skull and facial gland development. To delineate signaling pathways in which Foxc1 is involved we compared the transcriptomes of whole heads of Foxc1+/+ and Foxc1−/– embryos using a candidate cDNA array comprising genes expressed in the head mesenchyme and ocular region, and a 7K oligo array. Absence of Foxc1 led to downregulation of Stat1 and Galnt4, and upregulation of Tgfβ1i4 at embryonic day 13.5 in the developing head mesenchyme. Comparative analyses revealed differences in the expression pattern of Tgfβ1i4 in the head mesenchyme of Foxc1−/– and Foxc1+/+ embryos. In the ocular regions of Foxc1−/– embryos, Tgfβ1i4 was expressed in higher levels in the conjunctival epithelium and in the condensing mesenchyme on the nasal aspect of the developing eye while in wild‐type embryos more intense expression was seen in the mesenchyme on the temporal aspect of the eye. Such data indicate that Foxc1 regulation of Tgfβ1i4 is complex and may be cell‐type dependent. Analysis of the regulation of Tgfβ1i4 by Foxc1 in a more homogenous cell population, mesenchymal cells isolated from the periocular region revealed that, in these cells, Foxc1 negatively regulated Tgfβ1i4 expression, presumably via secreted factors such as TGF‐β1. Since Foxc1 expression is essential for normal craniofacial development, it is possible that its downstream targets play a role in the development of the phenotypes associated with null mutations in Foxc1.

The active fraction of plasmatic plasminogen activator inhibitor type 1 as a possible indicator of increased risk for metastatic melanoma

Plasminogen activator inhibitor type 1 (PAI1) is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key-enzyme in the metastasis and vascularization of solid tumors. The aim of this study was to determine whether high or low plasma levels and/or activity of PAI1 correlate with the presence of metastatic disease for patients with melanoma. We hypothesized that the presence of metastases could result in a disturbance of the fibrinolytic balance of the blood. To test our hypothesis, we have developed a unique enzyme-linked immunosorbent assay (ELISA) that can measure both the total amount as well as the active fraction of PAI1 in the plasma. We then used this novel assay to analyze the plasmatic PAI1 levels and activity of patients with advanced melanoma (AM, n = 18) and primary melanoma (PM, n = 21) and compare it to a control population (n = 38). We found no statistically significant difference in the total plasmatic PAI1 levels between the controls and patients with PM or AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). The difference between the control and AM groups was highly significant (P = 0.0042). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. Surprisingly, the difference between the control and PM groups was also significant--although borderline (P = 0.0488). Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity might be a biological marker of increased metastatic risk.

Molecular cloning and sequence analysis of a chicken cDNA encoding tyrosinase-related protein-2/DOPAchrome tautomerase

We have cloned and sequenced a chicken cDNA encoding an l-DOPAchrome tautomerase (DCT) from an embryonic melanocyte cDNA library. The chicken DCT gene encodes a deduced protein of 516 amino acids (aas) and shares 69.2% and 69.9% aa sequence identity with the deduced mouse and human DCT proteins, respectively. Northern blot hybridisation analysis reveals a DCT transcript of 3.5kb in RNA from the retinal pigment epithelium (RPE) of chick embryos. Genomic Southern blot hybridisation analysis suggests that the chicken DCT gene consists of several introns and spans between 15 and 30kb of the chicken genome. This study completes the sequencing of all the members of the chicken tyrosinase-related protein gene family and provides evidence that this gene family is conserved between avians and mammals.