Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.

Cutaneous responses to topical methyl nicotinate in black, oriental, and caucasian subjects.

The response of human skin to topical methyl nicotinate (MN) has been monitored in black, oriental, and caucasian subjects. The study aimed to address the question: "Do racial differences in percutaneous absorption and microcirculatory sensitivity exist?" MN-induced vasodilatation was assessed visually and by laser Doppler velocimetry (LDV). At three dose levels, in the three subject populations, four parameters were compared: (a) the diameter of the maximum visually perceptible erythematous area (Emx); (b) the area under the erythematous diameter versus time curve (AUE); (c) the maximum LDV response (Lmax); and (d) the area under the LDV response versus time curve (AUL). At p less than 0.05, AUL (black) greater than AUL (caucasian) for all MN concentrations; AUL (oriental) greater than AUL (caucasian) for the higher dose levels. Emx, AUE and Lmx showed no significant differences between races within concentrations. For all subjects, Emx, AUE, and AUL were significantly dependent on MN dose whereas Lmx was not. The results suggest that some racial differences in response to topical MN exist and that perception of these distinctions may depend upon the method of measurement.

The relation between cutaneous blood flow and cell content in the tuberculin reaction.

The relationship of velocity of blood flow to density and microanatomical distribution of inflammatory cells in the dermis was studied in 20 human tuberculin tests. Most positive reactions showed maximal blood flow velocities (measured as red blood cell (RBC) flux) at the centre of the reaction, but the two most intense responses showed 'central relative slowing' (CRS) with higher RBC flux at the periphery. Two of the four clinically negative reactions showed a considerable acceleration of blood flow, but the other two showed no such acceleration. The packing density of lymphocytes/monocytes in the perivascular zone was greater in the stronger positives than in the weaker reactions. The density of cells in the intervening dermis was markedly lower than in the foci: the lesions with CRS had the highest density of cells in the diffuse infiltrate of the reticular dermis. At the centre of the reaction, blood flow velocity was generally related to density of cellular infiltrate, except in those with CRS, which had a disproportionately lower blood flow velocity. The finding that the circulatory adaptation to a delayed hypersensitivity reaction can be inadequate may explain the dermal acidosis previously observed in intense skin test reactions, and may be the underlying mechanism of necrosis in hypersensitivity reactions.

Light and electron microscopy of fetal rabbit skin with special reference to the role of mesenchymal cells in epidermal differentiation.

Fetal rabbit skin between 10 and 26 days of gestation was observed by light and electron microscopy. The present study indicates that rapid epidermal differentiation, including the epidermal downgrowths as primordia of the hair follicles, is induced by aggregation of mesenchymal cells associated with growing capillaries beneath the epidermis. In addition, the transformation of these mesenchymal cells to vasoformative cells for rapid capillary growth is further evidenced by this study. Glycogen-storing cells in the periderm are most numerous between 15 and 18 days of gestation, but disappear almost completely by 20 days when a capillary network develops beneath the epidermis. This may imply an active involvement of the periderm in glucose uptake from the amniotic fluid during early developmental stages of the skin.

Oxygen tension and consumption measured by a tc-PO2 electrode on heated skin before and after epidermal stripping.

Oxygen tensions, cutaneous blood flow rate, and skin oxygen consumption rate were determined by tc-PO2 measurements at an electrode temperature of 45 degrees C. The epidermal surface was stripped by 50 applications of adhesive plaster to the surface. Ten healthy, normotensive adults were examined. Cutaneous blood flow rate was 41.2 +/- 8.6 ml X (100 g)-1 X min-1 before and 42.8 +/- 5.9 ml X (100 g)-1 X min-1 after epidermal stripping. Oxygen consumption before stripping was 0.327 +/- 0.065 ml O2 X (100 g)-1 X min-1, and after stripping it was determined at two different saturation levels to be 0.208 +/- 0.072 ml O2 X (100 g)-1 X min-1 and 0.251 +/- 0.096 ml O2 X (100 g)-1 X min-1. Capillary temperature was estimated to be approximately 43 degrees C before and after stripping. At this temperature mean arterial PO2 was estimated to be 18.1 kPa (136 mmHg), which would be reduced by the computed local metabolism to a mean capillary PO2 of 14.4 kPa (108 mmHg) before stripping and 15.2 kPa (114 mmHg) after. Stripping increased mean skin PO2 from 10.9 +/- 0.6 kPa (82.3 +/- 4.7 mmHg) to 14.6 +/- 1.0 kPa (109.4 +/- 7.7 mmHg). Thus, stripping eliminated 82% of the gradient between the capillaries and electrode while reducing the computed oxygen consumption by 23-36%. It is concluded that the epidermal membrane is a significant barrier to oxygen diffusion and that the transcutaneous oxygen electrode has a significant effect on skin PO2 owing to its own even low oxygen consumption. This will reduce the observed skin PO2 significantly.

Revascularization of reversed dermis grafts.

The process of vascularization and the vascular patterns in reversed dermis grafts (RDG) and full-thickness skin grafts (FTSG) in the rat were studied at various intervals after transplantation by a combination of microangiography and histologic techniques. In the early stages the RDG became vascularized more rapidly than the FTSG. However, the inflammatory reaction in the RDG persisted for longer periods than that in the FTSG in the later stages. The prolonged inflammatory reaction in the RDG was presumed to be due to the lack of epidermis for two to three weeks after surgery. Comparatively severe contraction of RDG may result from this prolonged inflammation and the delayed epithelialization.

Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis.

A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1 X 10(-9) M), isobutyl methylxanthine (3.3 X 10(-5) M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies.

[Influence of the stratum corneum on transepidermal water loss and Bioavailability of a new topical steroid, difluprednate: clinical implications (author's transl)]. / Influence du stratum corneum sur la perspiration insensible et sur la biodisponibilité d'un nouveau stéroïde topique, le difluprednate. Implications thérapeutiques.

Comparative study of experimental and theoretical curves obtained by plotting transepidermal water loss against stratum corneum thickness in man, shows that every layer in the stratum corneum acts as part of the epidermal barrier to water loss. Another function of the stratum corneum is to decrease the cutaneous penetration of topical corticosteroids like difluprednate and to modify their bioavailability ('corticosteroid reservoir'). Our data suggest that variations in stratum corneum thickness between subjects explain variation of transepidermal water loss and sensitivity difluprednate, as there is a close relationship between these two parameters. It is then conceivable than the phototype has clinical implications is there really exists a relationship between phototype, stratum corneum thickness and sensitivity to steroids.

The reversed dermis flap.

The reversed dermis principle of Hynes has been applied to flaps. There are 2 main varieties: the direct bridge pedicle flap and the turn-over local flap but the same principle might occasionally be useful in free flaps.