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Biomedical subjects

M A Karasek

Publications and source records attributed to M A Karasek.

At least 19 recordsLinked to original sources
PubMed1992

Histamine-modulated transdifferentiation of dermal microvascular endothelial cells.

Homeostatic and inflammatory functions of skin microvessels are tightly regulated by vasoactive amines. Following stimulation with histamine, dermal microvascular endothelial cells (MEC) undergo a rapid change in phenotype (transdifferentiation) and subsequently exhibit an enhanced rate of growth. To elucidate mechanisms regulating MEC transdifferentiation, this study investigated the functional relationships among vimentin, Ca2+, and protein kinase C (PKC) in histamine-modulated dermal MEC in vitro. Distribution of vimentin and PKC in foreskin-derived MEC cultivated in a modified Iscove's medium was assessed with immunocytochemistry. Calcium ion kinetics in histamine-treated MEC were analyzed using the Ca2+ probe Fluo-3 in conjunction with interactive laser cytometry. Histamine, acting through H-1 receptors, produces a rapid (less than 100 ms) and differential elevation of free calcium in each of three cytological compartments defined by the vimentin cytoskeleton in epithelial MEC. A distinctive compartmentalized and nonuniform distribution of PKC precisely coincides with that observed for free-Ca2+ released in response to histamine. The studies reveal that histamine modulation of the MEC phenotype is associated with a rapid patterned reorganization of the vimentin skeleton. It is hypothesized that histamine induces vimentin post-translational modifications by activating a spatially localized interaction among cytoplasmic free Ca2+, PKC, and the vimentin matrix. The results further suggest that vimentin, in addition to its structural role, may participate in signal transduction and gene regulation processes in effecting MEC transdifferentiation.

Calcium↗
PubMed1992

Factors affecting the growth and maintenance of human skin mast cells in cell culture.

The isolation and kinetics of survival of human mast cells from newborn and adult skin is described. Recombinant human interleukins and conditioned medium from several human cell lines were tested for their ability to maintain mast cells in vitro. Growth medium supplemented with IL-2, IL-4 and conditioned medium from a mixed lymphocyte culture enhanced mast cell survival resulting in a 30-fold increase in survival (relative to that obtained with non-supplemented medium) at 7 days, and a 15-fold increase at 15 days. Cell survival for time periods longer than 21 days was not observed. Inclusion of cAMP, agents that elevate cAMP, insulin, and epidermal growth factor in supplemented growth medium prevented the enhanced survival by 40-70%. Incorporation of bromodeoxyuridine (BrdU) into mast cells in 3-day cultures demonstrated that 15% of the mast cell population was capable of proliferation. At 21 days, no incorporation of BrdU could be detected. After 3 days in culture mast cells released 16% of their histamine stores in response to A23187 and 10% in response to anti-human IgE. Electron microscopy of cultured cells at 3 days revealed cells with both intact and empty mast cell granules. These results demonstrate that human skin mast cells proliferate in response to cytokines and release histamine when stimulated with classical secretagogues. Since human skin mast cells retain these basic properties in vitro, they may be useful in further functional studies involving their proliferation and secretion.

Adult↗
PubMed1992

Modification of proto-oncogene expression by phorbol esters in human dermal microvascular endothelial cells.

Following activation with the inflammatory mediator phorbol myristate acetate (PMA), human microvascular endothelial cells (DMEC) is olated from the human dermis (DMEC) rapidly and dramatically convert from a classical epithelioid morphology to a spindle-shaped configuration. This is accompanied by changes in the organization of gap junctions and the vimentin and actin cytoskeletons. This report describes the sequential changes in the expression of four proto-oncogenes, c-fos, c-myc, c-sis and H-ras in DMEC following PMA exposure. The synthesis of c-fos mRNA was transiently induced by PMA from a basal concentration below the limit of detection to a maximum at 60 min., declining to the unstimulated level within 2 hrs. Synthesis of c-myc mRNA declined continuously and reached 37% of control levels over 16 hrs. Expression of c-sis which encodes for the B chain of platelet-derived growth factor, also declined to 34% of the control value over 16 hrs. There was no change in the synthesis of H-ras mRNA nor of beta-actin mRNA which was used as a control. The expression of c-myc in normal DMEC was compared to a human dermal microvascular cell line transformed by SV-40 (TREND). The TREND cell line maintains a permanent spindle-shaped configuration under all growth conditions and multiplies faster than DMEC. In contrast to the non-transformed cell cultures, expression of c-myc in TREND cells was induced by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1991

Microvessel endothelial cell transdifferentiation: phenotypic characterization.

Human dermal microvessel endothelial cells (MEC) have two basic functions: maintenance of tissue homeostasis and facilitation of inflammatory responses. The former requires that the endothelium expresses traits of an epithelium, while inflammatory reactions are associated with intimal disruption. Acute inflammation transiently alters endothelium, whereas chronic inflammation may result in vessel reorganization and MEC mesenchymalization. Foreskin MEC in vitro undergo a similar epithelial-mesenchymal modulation. In the presence of cAMP, cultivated dermal MEC exhibit the structural and functional characteristics of an epithelium. MEC grown in cAMP-deficient medium initially have a "transitional" configuration and are subsequently transformed into mesenchymal cells. If cAMP is replaced by histamine, MEC maintain a stable intermediate transitional configuration. Transitional MEC refed cAMP-supplemented medium revert to an epithelial phenotype, whereas parallel cultures fed cAMP-deficient medium are transformed into mesenchymal cells. Phenotypic modulation can be induced without cell division and thus provides a unique example of direct transdifferentiation. Our data furthermore suggest that this transdifferentiation results in the acquisition of properties usually attributed to cells of the reticuloendothelial system.

Cell Differentiation↗
PubMed1991

[Recent findings in physiology and pathology of endothelial cells of the skin].

Perivascular cell infiltrates with concomitant changes of the endothelium represent a histological hallmark of numerous inflammatory dermatoses. Therefore, the pathophysiology of dermal endothelial cells is of particular importance for the understanding of inflammatory processes within the skin. In recent years, the development and refinement of endothelial cell culture techniques have facilitated investigations in cell biology and molecular biology, which complement histological observations, so leading to sounder knowledge of endothelial cell pathophysiology. The following review will discuss characteristic features of endothelial cells as well as in-vitro culture techniques. Focussing on endothelial cell interactions with leukocytes and with the extracellular matrix, recent developments in this area of research will be presented.

Cell Adhesion Molecules↗
PubMed1990

Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

1-Methyl-3-isobutylxanthine↗
PubMed1990

New developments in our understanding of the biology of psoriasis.

Increased keratinocyte proliferation and inflammation are two hallmarks of psoriasis. In this paper new developments in skin biology and biochemistry that help us to understand these two features of the disease are discussed. Methods to control proliferation and inflammation based on these scientific developments are presented.

Cyclosporins↗
PubMed1989

Microvascular endothelial cell culture.

Microvascular endothelial cells play a central role in inflammation, tumor metastasis, and wound healing. Methods to study these processes in vitro using cells isolated from adult skin, from the inner and the outer segments of the neontal foreskin, and from experimental animals are reviewed. A new modified Iscove's medium supplemented with 2% pre-partum maternal serum, dibutyryl cyclic AMP, isobutyl methylxanthine, thymidine, and hypoxanthine is described. This modified medium supports growth of both adult and neonatal endothelial cells up to seven passages with retention of cytologic markers closely identified with endothelial cells (Weibel-Palade bodies, Factor VIII-associated antigen). Several functions associated with the microvasculature in situ are expressed by microvascular endothelial cells in cell culture. Such functions include the formation of a basement membrane, angiogenesis, intercelluar gap formation in response to vasoactive agents, and the attachment and migration of lymphocytes through endothelial monolayers.

Adult↗
PubMed1988

Effect of neuropeptides present in skin on the proliferation of human peripheral blood mononuclear cells and T cells.

The release of neuropeptides, such as substance P (SP) and somatostatin (SOM), from primary sensory nerve fibers has been implicated in the modulation of local immune responses in surface tissues, such as the skin, the pulmonary airways, and the gastrointestinal mucosa. We have investigated the influence of six neuropeptides substance P (SP), somatostatin (SOM), substance K (SK), vasoactive intestinal peptide (VIP), bombesin (BOM), and adrenocorticotropic hormone (ACTH) on the proliferation of resting and partially stimulated human peripheral blood mononuclear leukocytes (PBMLs) and T lymphocytes. Neuropeptides in concentrations from 10(-7) to 10(-12) M were added to either resting or partially stimulated cells [interleukin-2 (IL-2), concanavalin A (Con A), and phytohemagglutinin (PHA)]. Cellular proliferation was assessed by incorporation of 3H-thymidine after 72 h. With the exception of SP, no significant effect of any of these neuropeptides on 3H-thymidine incorporation was found. In resting cells, 10(-9) MSP elicits an 80...maximal increase of 3H-thymidine incorporation, whereas no statistically significant effect on partially stimulated leukocytes was found. These results contradict a previous report on a significant mitogenic effect of SP on partially stimulated T cells. Considering the very minimal effect of SP on resting cells and, particularly, the absence of an effect on partially stimulated cells, we would question a significant modulatory role for SP and the five other neuropeptides in the proliferation of immunocompetent cells in skin.

Humans↗
PubMed1988

Leukotriene B4 enhances adherence of human polymorphonuclear leukocytes to dermal microvascular endothelial cells in vitro.

Adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells (ECs) is a crucial step in the diapedesis of inflammatory cells to the site of inflammation. We have demonstrated that leukotriene B4 (LTB4), a metabolite of the arachidonic acid cascade, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) significantly enhance the binding of human PMNs to selected populations of human dermal microvascular endothelial cells (MECs) in vitro. MECs were isolated from the vascular-rich portion of foreskins of newborns. MECs were grown in Iscove's modified Dulbecco's media with 2% prepartum serum and 8% newborn calf serum on 1% gelatin-coated plastic dishes. PMNs isolated from five human donors were added to the culture dishes for varying time intervals (usually 30 min) in the presence and absence of the chemotactic stimuli LTB4 and FMLP. Addition of PMNs to MECs in the absence of chemotactic stimuli results in "baseline" binding to the MEC monolayer. About one in every 150 ECs binds more than five PMNs. These selected ECs are randomly distributed throughout the monolayer. LTB4 from 10(-10) to 10(-7) M increases the number of MECs which selectively bind PMNs by 260% at 10(-7) M. FMLP also increases adherence in qualitatively and quantitatively similar fashion. These data support a role for LTB4 in the mediation of adherence of neutrophils to dermal MECs. In contrast to other endothelial cells from the large blood vessels, such as from umbilical veins or calf thoracic aortae, PMNs bind only to selected MECs in culture, even when stimulated with LTB4 or FMLP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed1988

Preferential binding of monocytes and Leu 2+ T lymphocytes to interferon-gamma treated cultured skin endothelial cells and keratinocytes.

Recombinant gamma interferon (r-IFN-gamma) increases the adherence of peripheral blood mononuclear leukocytes (PBMLs) to cultured keratinocytes and cutaneous microvascular endothelial cells (MECs). To determine which specific type of PBMLs bound to these r-IFN-gamma treated cells, we performed immunophenotyping on the adherent PBMLs. The adherent PBMLs were detached from the r-IFN-gamma treated keratinocytes and MECs by adding EDTA, and collected by cytocentrifugation, followed by immunocytochemical staining using a panel of monoclonal antibodies. Our results reveal that the relative adherent population of PBMLs was composed of approximately 60%-70% monocytes and 18%-24% Leu 2+ T lymphocytes (T-cytotoxic/suppressor) which preferentially bound to r-IFN-gamma treated keratinocytes and MECs. There was some lesser binding by Leu 3 + lymphocytes (T-helper/inducer); approximately 8%, and no binding of B lymphocytes. Since r-IFN-gamma also induced HLA-DR expression in keratinocytes and MECs, these in vitro data suggest that r-IFN-gamma may play an important role in the immunobiology of diverse skin diseases such as graft vs host disease, lichen planus, and other inflammatory dermatoses, because the keratinocytes express HLA-DR and the predominant T-cell subset in the epidermis is Leu 2 + (over the Leu 3 + T cell) in all of these conditions. These results represent a direct attempt to explain in situ immunophenotypic mononuclear leukocyte subset distribution patterns by using r-IFN-gamma and purified cultured cells such as keratinocytes and MECs. We propose that IFN-gamma, by both increasing the adherence of PBMLs, and promoting selective binding of monocytes and Leu 2 + T lymphocytes to both keratinocytes and MECs, may be important in regulating PBML localization and recirculation in the skin.

Antibodies, Monoclonal↗
PubMed1987

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.

Antibodies, Monoclonal↗
PubMed1986

Synthesis of basement membrane-specific macromolecules by cultured human microvascular endothelial cells isolated from skin of diabetic and nondiabetic subjects.

Microvascular endothelial cells isolated from abdominal skin of diabetic and nondiabetic adults were maintained in culture by serial passage. Both cell types showed typical endothelial cell morphology, expressed factor VII-associated antigen, contained Weibel-Palade bodies, and produced an extensive subendothelial extracellular matrix containing type IV (basement membrane) procollagen. Biosynthetic studies using radioactive amino acids indicated that under the conditions of cell culture the matrix proteins newly synthesized by MEC of both cell types were similar in type and amount. Both cell types produced type IV procollagen, laminin, and fibronectin, which were deposited in the matrix. Electron microscopy showed that the matrices of both cell types had a similar multilayered, discontinuous, filamentous ultrastructure. Immunoperoxidase staining showed type IV collagen to be distributed similarly, in a fibrillar meshwork, in both matrices. The extractability of individual matrix macromolecules from both matrices was identical; 4 M urea or guanidine-HCl partially removed fibronectin and thrombospondin, but reducing agent was required to solubilize type IV procollagen. The results suggest that diabetic microangiopathy is not due to an inherent defect in the endothelium, and that this in vitro system may be useful for examining environmental factors possibly involved in its development.

Aged↗
PubMed1986

Differential effects of 5-fluorouracil on human skin melanocytes and malignant melanoma cells in vitro.

We have observed differential effects of the cytotoxicity of 5-fluorouracil (5-FU) on human skin melanocytes and malignant melanoma cells in vitro. In the absence of 5-FU the melanoma cells multiplied much more rapidly than the melanocytes. With a 7-day exposure of 5-FU (1.92 X 10(-5) to 3.84 X 10(-4) M) all the melanoma cells died by 5 W, while even two times longer exposure (a 14-day exposure) of 5-FU the melanocytes survived till 6 W and increased at the lower concentration. In the presence of 5-FU there was a different compensatory increase in [3H] thymidine incorporation between the two cell types. That is, compared with the large increase observed in the incorporation by the melanoma cells, a small increase in the incorporation by the melanocytes was observed. The reason for these differential effects of 5-FU may be the difference in the cell cycle and the post-injury cell renewal of the two cell types.

Cell Division↗
PubMed1985

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane.

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.

Basement Membrane↗
PubMed1985

Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin.

We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.

Aging↗
PubMed1985

A new method for obtaining scanning electron microscopic images of the reorganization process of human dermal microvascular endothelium in vitro.

A new method for obtaining scanning electron microscopic images of the reorganization process of endothelial cells has been developed. When covered with a collagen-coated disk, all the cultured endothelial cells reorganized on the collagen of the disk, which was easily taken out from the dish to process for SEM. The reorganization process could be divided into four stages: endothelial cell growth (Stage 1), reticular network formation (Stage 2), tubular structure formation (Stage 3), and cytolysis of the tube (Stage 4). Between Stages 1 and 2 the endothelial cells transformed from a cobblestone to a spindle-shaped pattern and fused each other, forming a board-like structure. Between Stages 2 and 3 break up of parts of the board-like structure and outflow of a necrotic mass from the centre of the structure occur. At Stage 3 a tubular structure is formed following enwrapping of the cleared centre by the surrounding endothelial cells. This method produces a means to study the angiogenesis in a variety of disorders including tumours and wound-healing process using SEM.

Cells, Cultured↗