Marvin A. Karasek

Prostaglandin I2 is not a major metabolite of arachidonic acid in cultured endothelial cells from human foreskin microvessels.

Prostaglandin I2 (PGI2), a potent vasodilator and inhibitor of platelet aggregation, is a major product of arachidonic acid metabolism in endothelial cells that are derived from large blood vessels (e.g., umbilical veins). We have examined whether PGI2 is also a major product of arachidonic acid metabolism in cultured endothelial cells that are derived from dermal microvessels in human newborn foreskin. Supernatants from confluent monolayers of endothelial cells that had been incubated for 20 min with [3H]arachidonic acid and the calcium ionophore A23187 (10 microM) were assayed for prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha (PGF1 alpha) (the stable metabolite of PGI2) by using authentic standards and high performance liquid chromatography. Whereas supernates from stimulated umbilical vein endothelial cells contained 6-keto-PGF 1 alpha much greater than PGF 2 alpha much greater than PGE2, supernates from stimulated foreskin microvessel endothelial cells contained PGF 2 alpha congruent to PGE2 much greater than 6-keto-PGF 1 alpha. Similar results were obtained when supernates from stimulated, unlabeled endothelial cells were analyzed by radioimmunoassay. These data indicate that PGI2 is not a major metabolite of arachidonic acid in cultured endothelial cells from human foreskin microvessels.

A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin

SummaryWhen multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin.To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions.To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads.To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS).Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.

Hereditary Absence of Sebaceous Glands in the Mouse

An autosomal recessive mutation, characterized by an absence of sebaceous glands, and by hyperkeratosis, alopecia, and single (rather than the usual multiple) hair-follicle units, has occurred spontaneously in the BALB/c strain of mouse. Studies in which reciprocal transplantations of skin were made between normal and mutant mice suggest that some diffusible substance(s) synthesized by normal skin can stimulate hair growth and alleviate the hyperkeratosis characteristic of the skin syndrome.

Inflammatory cytokines induce the transformation of human dermal microvascular endothelial cells into myofibroblasts: a potential role in skin fibrogenesis

Background:  The myofibroblast plays a central role in wound contraction and in the pathology of fibrosis. The origin(s) of this important cell type in skin has not been firmly established.

Interleukin‐1 induces major phenotypic changes in human skin microvascular endothelial cells

To determine the role of the pleiotropic cytokine interleukin‐1 (IL‐1) on the activation of endothelial cells during inflammation and angiogenesis, pure populations of human dermal microvascular endothelial cells (HDMEC) were obtained by immunoaffinity purification using Ulex europaeus agglutinin‐1 and platelet endothelial cell adhesion molecule‐1 (PECAM‐1) antibody. Exposure of HDMEC to IL‐1β induced morphologic and physiologic changes characterized by (1) phenotypic modulation of endothelial cells from epithelioid to spindle‐shaped cells accompanied by reorganization of vimentin filaments; (2) gradual decrease to a complete absence of the endothelial cell markers von Willebrand factor (vWf) and PECAM‐1; and (3) increased capability to form tubule‐like structures when overlaid with collagen gels. The IL‐1 effect on cell morphology, growth, and decrease of endothelial cell antigens was potentiated by basic fibroblast growth factor (bFGF). Similar results were observed in mitotically arrested γ‐irradiated cells demonstrating that the spindle‐shaped cells observed after IL‐1 stimulation were derived from epithelioid endothelial cells and that DNA synthesis was not required to effect these changes. Immunostaining with an antibody specific for human fibroblasts was negative and further confirmed the endothelial cell origin of the spindle‐shaped cells. These data demonstrate that IL‐1 can induce phenotypic changes in HDMEC from epithelioid to spindle‐shaped, mesenchymal‐like cells, that these cells are more susceptible to stimulation by bFGF, and that they lose biochemical and functional properties characteristic of epithelioid HDMEC. J. Cell. Physiol. 173:84–92, 1997.

ISOLATION AND LONG-TERM SERIAL CULTIVATION OF ENDOTHELIAL CELLS FROM THE MICROVESSELS OF THE ADULT HUMAN DERMIS

SumamryA 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 (1×10−9M), isobutyl methylxanthine (3.3×10−5M), 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.

Growth characteristics of human epidermal kerationcytes from newborn foreskin in primary and serial cultures

SummaryUsing gels of acid-soluble, collagen as a culture surface, trypsin-released keartinocytes from 0.1-mm, split-thickness sections of newborn foreskin may be plated with high efficiency and subcultured at a 1∶5 split a 2- to 3-week intervals for three subpassages. When plated at a density of 3.2×104 cells per cm2, keratinocytes attach to the gel with an efficiency of over 70%; after a lag phase of 3 days, the cells multiply exponentially with a doubling time of 60 hr. Cultures reach a growth-plateau phase at a density of 47.7×104 cells per cm2. Both hydrocortisone and epidermal growth factor (EGF) stimulate slightly the growth of primary cultures; both factors are required for proliferation of the 2nd and further passage of keratinocytes. As the cultures reach, confluence multilayers, of stratified cells are formed and cells of squamous morphology are spontaneously released from the surface. When the released cells and the attached cells are pulsed with [3H]-histidine and [14C]-leucine, a higher ratio of histidine to leucine is observed in the released cells indicating the biochemical onset of maturation. Orange G-Aniline Blue staining of the released cells show some of the cells to be completely keratinized. Fibrous proteins extracted from the cultured cells and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis display the characteristic stratum corneum proteins of 60,000 and 66,000 daltons.

Phenotypic and functional characterization of lymphocytes that bind human microvascular endothelial cells in vitro. Evidence for preferential binding of natural killer cells.

The microvascular endothelium has been postulated to be a critical target in the rejection of vascularized allografts. This study was undertaken to examine the ability of human sheep erythrocyte rosette forming lymphocytes (E-RFC) to form stable conjugates with microvascular endothelial cells (EC), and to assess whether a receptor-ligand interaction mediates this event. Human foreskin microvascular EC monolayers were used as targets of chromium-51-labeled E-RFC in a quantitative adherence assay. Binding was saturable, displaceable by unlabeled E-RFC, augmented by recombinant interleukin 1 (rIL-1) and inhibited by anti-LFA1 antibody. The Leu-11+ lymphocyte subset, known to be enriched for natural killer (NK) cells, bound preferentially. Only the EC-adherent lymphocyte fraction contained NK effectors, which lysed EC and classical NK targets. Thus, NK cells adhere to microvascular EC via a specific receptor-ligand interaction. The possibility exists that such binding occurs in recipients of vascularized allografts, representing the initial stage of graft rejection.

In vitro characteristics of neonatal hemangioma endothelial cells : Similarities and differences between normal neonatal and fetal endothelial cells

Background: Increased angiogenesis and eventual involution are major characteristics of neonatal hemangiomas. The mechanism to explain this transition is not completely understood.

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro.

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase.