Melanoma x macrophage fusion hybrids acquire increased melanogenesis and metastatic potential: altered N-glycosylation as an underlying mechanism.

We recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential. With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content and increased responsiveness to MSH compared to parental cells. Here we investigated the hybrid melanotic phenotype in more detail, comparing the pigmentary systems of hybrids and parental Cloudman S91 cells by several techniques. Cells were studied by electron microscopy, cell lysates were analyzed for tyrosinase (E.C.1.14.18.1) activity, and melanosomal proteins were analyzed by gel electrophoresis and immunoblotting. Melanosomes in parental Cloudman melanoma cells were few in number and relatively amorphous, whereas those in the hybrids were numerous and heavily pigmented, containing highly organized lattice structures. Both basal and MSH-inducible tyrosinase activities were elevated several fold in hybrids compared to parental cells. Tyrosinase, TRP-2, and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages, which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. By using 3H-glucosamine as a marker of N-glycosylation, its incorporation into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors, and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.

Topographic mapping of the cutaneous microcirculation using two outputs of laser-Doppler flowmetry: flux and the concentration of moving blood cells.

Using a "needle" probe in a template probe holder, we measured the flux and the concentration of moving red blood cell (CMBC) outputs from a Perimed (PF2B) laser-Doppler instrument at 1-mm2 contiguous sites in a 8 x 8-mm area on the flexor forearm of three subjects. Using the means of the flux and CMBC recorded at each spot, a topographic contour map was constructed for each of these parameters. Viewing the two maps together, sites with four different combinations of flux and CMBC could be identified. Trephine biopsies (2 mm) of three representative sites in each subject were performed and the upper plexus was reconstructed in 3 dimensions from serial sections. High flux/high-to-medium CMBC sites were found over the spot where the ascending arterioles entered the upper plexus. Medium flux/medium-to-low CMBC sites and low flux/medium CMBC sites were found in the peripheral part of the vascular unit that was fed by the ascending arteriole. The low flux/low CMBC sites were relatively avascular zones. Video imaging of the upper plexus in the forearm showed the same overall vascular pattern as the contour maps. The highest flux and CMBC signals were recorded when horizontally oriented vessels were present in the upper third of the plexus (400-650 microns below the stratum corneum). Topographic mapping will allow one to selectively identify different microvascular areas in the skin for physiological studies.

Three-dimensional reconstruction of endothelial cell gaps in psoriatic vessels and their morphologic identity with gaps produced by the intradermal injection of histamine.

Endothelial cell gaps in psoriatic vessels and histamine-induced gaps in forearm skin of normal controls were reconstructed in 3 dimensions by a computer graphics system. The gaps in psoriatic vessels were present within the cell, at the intercellular junction, or concurrently at both sites. Histamine-induced gaps were found at the intercellular junction or at both intracellular and intercellular locations. The gaps were linear to oval and often contained cytoplasmic processes from one of the endothelial cells, suggesting that gap formation represents a cellular injury rather than a purely physiologic reversible phenomenon.

A study of the veil cells around normal, diabetic, and aged cutaneous microvessels.

The veil cells around normal, diabetic, and aged vessels were reconstructed in 3 dimensions by a computer graphics system from 120-140 serial ultrathin sections. The normal vessel was surrounded by a single layer of veil cells which had a wrinkled and pleated surface. The diabetic vessels were surrounded by 3-6 layers of cellular material produced by increased numbers of veil cells and their associated cytoplasmic sheets. The veil cells around aged vessels appeared to have the same length as young and diabetic veil cells but were underdeveloped in their lateral extensions so that they did not cover the vessel circumferentially as well as did the normal veil cells. Preliminary data suggest that young, diabetic, and aged veil cells have the same metabolic activity per unit area of cytoplasm and are of the same length. The abnormal thickness or thinness of the vascular wall of dermal microvessels appears to be related to the degree of development and numbers of veil cells around the vessels rather than any change in their basic metabolic activity from normal.

Ultrastructural abnormalities of the microvasculature and elastic fibers in the skin of juvenile diabetics.

We examined by electron microscopy the calf and buttock skin of 15 juvenile diabetics, 9-36 years old, to determine whether microangiopathy was present. We found in 14 of 15 buttock specimens, and in all 15 calf skin specimens, that the vascular walls were thickened by the peripheral deposition of a belt of basement membrane-like material admixed with variable amounts of collagen (reticulin) fibrils. In addition there appeared to be an increased amount of unidentified material, most likely basement membrane, deposited within the vascular wall itself. We also found age-related elastic fiber abnormalities in the dermis and in the arterioles which were identical to those observed in normal, nondiabetic aged individuals 50-93 years old. We suggest that the vascular and elastic fiber abnormalities in diabetic skin may represent a manifestation of accelerated aging rather than being related to the various underlying metabolic derangements in diabetes.

Ultrastructure of the human dermal microcirculation. IV. Valve-containing collecting veins at the dermal-subcutaneous junction.

Valve-containing collecting veins 70-120 micron in diameter were found at the interface of the dermis and fat. Two cusped valves with sinuses were found at most points where 25- to 50-micron venules entered these larger veins as well as in the lumens of the larger veins unrelated to branch points. The walls of these valve-containing veins in their narrowest portions were composed of smooth muscle cells, collagen fibers, and homogeneous-appearing basement membrane material. As the vessel became wider, elastic fibers first appeared just inside the most peripheral layer of smooth muscle cells, and gradually occupied the spaces between all the layers of smooth muscle cells. However the subendothelial zone was frequently devoid of elastic fibers. The valves were always pointed in the direction of the larger vessel as would be expected in a collecting vein. Recent experiments by other workers have demonstrated that the capillary blood flow in human nail-fold capillaries is pulsatile, thereby providing physiologic reasons for the presence of valve-containing veins at the dermis-fat interface.

Ultrastructure of the human dermal microcirculation. III. The vessels in the mid- and lower dermis and subcutaneous fat.

This paper describes the ultrastructure of the microcirculatory vessels in the mid- and lower dermis and subcutaneous fat. Reconstruction of vessel walls, tracing out the courses of individual vessels, and survey examination of vessels were carried out by various combinations of routine and serial, ultrathin, and 1-micron sections. Intracellular myofilamentous bundles associated with extracellular filaments were a characteristic feature of the endothelial cells in arterioles possessing an internal elastic lamina, but were only rarely seen in endothelial cells of venules. The ultrastructural features of these bundles and filaments suggested both contractile and anchoring functions. The elastic lamina of the arterioles was shown to be composed of individual elastic fibers oriented in the long axis of the blood vessel under the endothelium, rather than being a continuous sheet as in larger arteries and arterioles. The capillaries in the fat had walls of normal thickness (0.1-0.3 micron) similar to capillaries in other organs, in contrast to the dermal capillaries whose walls are 2-3 micron thick. The arterioles, capillaries, and venules in the fat were frequently devoid of veil cells in contrast to those in the dermis. Venous capillaries with bridged fenestrations were found in close proximity to eccrine sweat glands and hair bulbs in the forearm, trunk, and buttock skin.