EXPERIMENTAL

CELL

RESEARCH

199,279-291

Histamine-Modulated

(1992)

Transdifferentiation of Dermal Microvascular Endothelial Cells’

BRUCE H. LIPTON,~KLAUSG.BENSCH,*ANDMARVIN Departments

of Dermatology

and *Pathology,

Stanford

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 Ca”, 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-l receptors, produces a rapid (cl00 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 Cazf, 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 o 1992 Academic press, IUC. MEC transdifferentiation.

INTRODUCTION Homeostatic and inflammatory blood components are mixed and essentially confined in a “closed” circulatory system ready to promptly respond to environmen-

1 This work was supported by Grants AR39470 and AR07422 from the National Institutes of Health and by Grant N0014-89-J-1098 from the Office of Naval Research. ’ To whom reprint requests should be addressed.

University

A. KARASEK

School of Medicine,

Stanford,

California

94305

tal needs and challenges. Selective activation of the two different blood functions is ultimately controlled by phenotypic modulations of the microvascular endothelial cells [l]. Under normal physiologic conditions, the endothelial lining of microvessels is composed of a continuous simple squamous epithelium [2, 31. Epithelial microvascular endothelial cells (MEC) express properties of a selectively permeable barrier and directly regulate homeostatic exchanges between the blood and surrounding tissues [4, 51. In response to injury or to inflammatory signals, MEC undergo a rapid transition in phenotypic appearance [l, 6, 71. Acute inflammatory responses, generally transient in nature, are characteristically associated with focal disruptions of the MEC epithelium [B-lo]. Chronic inflammation often leads to a complete disruption and reorganization of the intimal lining [ 111. Under these conditions epithelial MEC may acquire mesenchyma1 traits and contribute to granulation tissue formation and other repair processes [12, 131. Observations on tissue biopsies and in vitro experimental models have shown that inflammation-activated MEC may express fibroblast, smooth muscle, and macrophage-like phenotypic properties [l, 6, 7, 141. A rapid phenotypic modulation of the microvessel intima in response to inflammatory stimuli is critical for survival. Dermal microvessel endothelial cells are especially adept at switching from a homeostatic epithelial to an inflammatory transitional phenotype [l, 81. The phenotypic modulations of dermal MEC represent an example of transdifferentiation. In transdifferentiation, phenotypic transitions are regulated by a dynamic balance among components of the nucleus, cytoplasm, and extracellular environment [l, 15,161. Factors in the induction of transdifferentiation in MEC cultures derived from human foreskin have recently been described [l]. Histamine, a principal mediator of MEC physiology, alters microvessel structure and function [8, 17, 181. MEC, both in vivo and in vitro, express a biphasic response to histamine. First, histamine induces MEC morphological changes that are effected by H-l receptormediated calcium signalling [ 10, 19, 201. Initial calcium responses are associated with ion mobilization from in-

279

0014-4827/92

$3.00

Copyright 0 1992 by Academic Press, Inc. All

rights

of reproduction

in any

form

reserved.

280

LIPTON,

BENSCH,

ternal stores (e.g., endoplasmic reticulum, mitochondria) [Zl, 221. Sustained histamine responses are dependent upon transmembrane influxes of extracellular calcium [ZO, 22,231. Secondary to its effect upon phenotypic expression, histamine also stimulates MEC proliferation [ 1, 241. To further elucidate mechanisms regulating endothelial transdifferentiation and changes in gene expression, we report now on the molecular anatomy of histamineinduced MEC alterations. The investigations reveal that a patterned restructuring of the vimentin skeleton coincides with an inflammatory modulation of endothelial phenotypes. Vimentin matrix reorganization is initiated immediately following the appearance of a compartmentalized free Ca2+ response induced by histamine. Protein kinase C (PKC) distribution spatially coincides with the vimentin matrix and the histamineassociated free Ca’+. The established role of PKC in vimentin post-translational modifications suggests a causal relationship among Ca’+, PKC, and vimentin in histamine-induced MEC transdifferentiation. METHODS Dermal microvascular endothelial cells Epithelial MEC cultures. were isolated from neonatal human foreskin and grown on gelatincoated substrates in a modified lscove’s growth medium supplemented with 8% newborn calf serum, 2% prepartum maternal serum, 5 X 10e4 M dibutyryl CAMP (db-CAMP), 3.3 X 1O-5 A4 isobutylmethylxanthine (IMX), 1 X 10m4M hypoxanthine, and 1.5 X 1O-5 M thymidine [25]. MEC maintained in this medium express structural traits closely resembling endothelial cells in vivo, including Factor VIII antigen, Weibel-Palade bodies, tight junctions, and pinocytotic vesicles [25, 261. Cell growth and morphology were monitored with phase microscopy. Transitional and mesenchymal MEC cultures. Transitional and spindle-shaped MEC were obtained by replacing the standard growth medium with medium deficient in db-CAMP and IMX [27]. Cells expressing a transitional phenotype first become evident approximately 5 h after standard growth medium is replaced by CAMP-free medium. Spindle-shaped mesenchymal MEC generally appear after 3 days growth in CAMP-deficient medium [25, 271. Histamine-treated MEC cultures. Replicate cultures derived from individual foreskins were cultivated in various concentrations of histamine dichloride (Sigma). Two days after seeding in db-CAMP-supplemented growth medium, MEC cultures were refed with db-CAMPfree medium containing histamine in increasing concentrations ranging from lo-’ to 10e3 M. After 7 days, cell populations were quantified with a fluorometric assay for intracellular hexosaminidase [28]. The accuracy of the hexosaminidase assay was verified by counting samples of trypsinized cell suspensions in a hemocytometer. Morphologic and behavioral responses of MEC to histamine were observed in cultures by phase microscopy. Epithelial MEC cultivated in db-CAMP-supplemented medium, at various stages of confluence, were refed with db-CAMP-deficient medium 45 min to 1 h prior to histamine exposure. The medium was then aspirated and cultures were refed with 10d5 M histamine in db-CAMP-supplemented or -deficient media. Control and histamine-treated MEC cultures were fixed at selected times for either electron microscopy or immunofluorescence assays. Calcium dynamics. Changes in cytoslic free calcium were visualized using a fluorescent probe in conjunction with laser cytometry

AND

KARASEK

1291. Cell cultures were prelabeled with 5 pg/ml of Fluo-3 (Molecular Probes) in standard db-CAMP-supplemented medium or in dbCAMP- and IMX-deficient lscove’s medium containing 8% newborn calf and 2% human prepartum sera. Cultures were incubated for 45 min in a 95% air 5% CO, atmosphere at 36.5”C. At To, cell images were recorded and their areas digitized using an ACAS 570 interactive digital laser cytometer equipped with confocal microscopy and kinetic analysis program (Meridian Instruments, Ml). Histamine (10m5M) in balanced salt solution was added 10 s prior to the second scan. Up to 10 scans at l-min intervals were made through the course of each observation. Regional changes in free calcium were digitally assessed and tabulated. Histamine receptor analyses. Histamine receptor agonists and antagonists were employed to identify which histamine receptors were active in eliciting the observed morphological and mitogenic responses in cultivated MEC. As in the concentration studies above, selected histamine agonists (2.methylhistamine and 4-methylhistamine, a generous gift from T. M. Hollis) at a concentration of 10m5M in db-CAMP-free medium were added to replicate cultures. Cell populations were determined after 7 additional days in culture. Similarly, MEC cultures were exposed to histamine antagonists (5 X 10m5 M cimetidine and 5 X low5 M diphenhydramine, Sigma) in db-CAMPfree medium, which was immediately followed by the addition of lo-” M histamine to the medium. Cultivated MEC were fixed with 3% glutarElectron microscopy. aldehyde, postfixed in buffered 1% OsO,, stained with uranyl and lead salts, and examined in an Elmiskop 101 electron microscope

U, 261. Fluorescence assays. For cytoskeletal studies, cell cultures were fixed with 2.5% paraformaldehyde in PBS and subsequently permeabilized with 0.5% Triton X-100 in PBS. Cells were stained with mouse monoclonal antibodies to vimentin (Clone-V9, Boehringer-Mannheim) according to the manufacturer’s specifications. Primary antibodies were visualized with complementary FITC-conjugated antiglobulins (Dakopatts) in glycerol-mounted specimens, and examined in a Nikon Microphot FX fluorescence microscope. Antibody target specificity was assessed by quenching cells with autologous serum and observing nonspecific binding with FITC-conjugated antisera. In addition, preincubation of MEC with autologous serum was used to quench nonspecific binding prior to labeling with monoclonal antibodies. Similarly, the presence of protein kinase C was determined using FITC-conjugated monoclonal antibodies against an epitope common to both (Y and p forms of bovine brain protein kinase C (Amersham). Distribution and relative concentrations of cellular PKC were assessed with the ACAS 570 interactive digital laser cytometer employing confocal optics.

RESULTS Histamine

Modulation

of Dermal MEC

Histamine induces a biphasic response in cultivated MEC. The initial effect is a rapid structural modulation of MEC, followed by a second phase in which cells demonstrate an increased rate of mitosis. Histamine, in concentrations ranging from 1O-6 to lop3 M, produced no observable morphological effects upon epithelial MEC grown in medium containing 5 X 10m4M db-CAMP (Figs. 1A and 1B). However, if parallel cultures are exposed to medium deficient in db-CAMP for 1 h prior to treatment, histamine will induce an immediate remodeling of cultivated MEC. Morphological

HISTAMINE

MODULATION

changes induced by histamine in the absence of dbCAMP are illustrated in Fig. 1D. Intercellular gaps appear between adjacent epithelial cells as early as 30 s following histamine presentation. During the next 5 to 15 min, these gaps enlarge as individual MEC centripetally retract their attenuated peripheral margins (Fig. 1D). Although it is difficult to distinguish between a contraction and a process of cell detachment, the following morphological observations suggest that histamine induces cytoplasmic contraction in MEC: (a) MEC frequently maintain peripheral adhesive sites and develop retraction spikes between points of attachment and the condensing cell body (Fig. 1D); (b) MEC surfaces exhibit cytoplasmic blebbing resembling the contraction response of smooth muscle myocytes (Fig. lD, inset); and (c) the regularity with which polygonal MEC convert into bipolar cells, maintaining specific anterior and posterior binding sites, argues against a random detachment (Fig. 1D). Continued loss of intercellular junctions and cell contraction lead to a profound disruption of the epithelial monolayer. The number of histamine-sensitive epithelial MEC may vary among primary cultures, although the percentage of responsive cells (50-90%) is relatively uniform in replicate cultures derived from the same primary. Within a given field, individual cells and/or small clonal clusters may express a contractile response to histamine (Fig. lD, inset). MEC exhibiting a transitional or spindle-shaped mesenchymal phenotype do not express a morphological response upon presentation of histamine. The MEC contractile response is generally complete within 5 min of histamine addition, although morphological changes may be observed for up to 15 min. After this time, contracted cells relax and slowly reflatten. MEC maintained in histamine acquire a flattened bipolar transitional phenotype [l] and subsequently undergo mitoses. MEC in histamine-supplemented medium show increased mitotic activity compared to parallel control cultures. Changes in population dynamics of MEC exposed to increasing histamine concentrations, ranging from 10e6 to 10e3 M, are presented in Fig. 2. Histamine at 10m5M induces up to a 400% increase in cell numbers over cultures grown in db-CAMP-deficient and histamine-free growth medium. Although histamine does not induce a morphological response in db-CAMP-supplemented cultures, it will function as a growth factor and augment the established growth promoting influence of db-CAMP. Histamine can increase the rate of MEC proliferation in db-CAMPstimulated cultures by 30 to 50% (Fig. 3). The mitogenic effect is seen consistently, while absolute levels vary among different primary cultures. MEC in histamine-treated cultures subsequently refed with standard db-CAMP-supplemented growth

OF DERMAL

281

MEC

medium revert to the squamous epithelial morphology. However, if cells growing in a histamine-containing, dbCAMP-free medium are put into a medium lacking both db-CAMP and histamine, MEC irreversibly acquire a spindle-shaped mesenchymal configuration [l, 25, 271. Histamine

Receptors

Figure 3 compares the influence of histamine, 2methylhistamine (H-l agonist), 4-methylhistamine (H2 agonist), diphenhydramine (H-l antagonist), and cimetidine (H-2 antagonist) on MEC proliferation. Stimulation of cell proliferation by 2-methylhistamine and the concomitant inhibition of histamine-induced proliferation by diphenhydramine indicate that MEC respond to a calcium-associated H-l receptor stimulation. In contrast, the reduced response to 4-methylhistamine and the inability of the antagonist cimetidine to block the histamine response suggest that H-2 receptors do not play a significant role in the observed dermal MEC reactions in vitro. Histamine receptors on cultivated MEC were visualized with laser cytometry (Fig. 4) following treatment of the cells with 10m5M FITC-conjugated histamine (Molecular Probes). Confocal microscopy revealed that labeled receptors were clustered on the apical surface. Pretreating MEC with excess 1O-4 M unlabeled histamine or 10e4 M diphenhydramine blocked the binding of labeled histamine (not shown). Histamine

and the MEC Vimentin

Cytoskeleton

Each of the cultivated MEC phenotypes (epithelial, transitional, and mesenchymal) has a differently patterned vimentin cytoskeleton [ 11. The vimentin matrix in epithelial MEC subdivides cells into three concentric and radially symmetric zones (Figs. 5A and 5C). Centrally, vimentin encapsulates and compartmentalizes the nucleus within a filamentous sheath that is tightly anchored to the nuclear envelope (Figs. 5C and 6A). In turn, the nucleus and most membranous organelles are enveloped within a flattened spheroidal perinuclear compartment defined by a coarse vimentin reticulum (Fig. 5C). Isolated mitochondria and cisterns of endoplasmic reticulum are frequently enmeshed within the fibrous vimentin reticulum (Figs. 6B-6D). A dense peripheral bundle of vimentin encircling the reticulum binds to the plasmalemma and physically delimits the central organellar-rich region of MEC from the cell’s attenuated peripheral margins (not shown, see Fig. 2B in 111). The vimentin cytoskeleton of epithelial MEC undergoes a rapid reorganization upon exposure to histamine. Sites of junctional dissolution and cell contraction constitute the first changes observable with the light microscope (Fig. 1D). The vimentin skeleton begins to depolymerize within 60 s of histamine stimulation (Fig. 5B).

282

LIPTON,

BENSCH,

AND

KARASEK

FIG. 1. Morphological response of cultivated dermal MEC to histamine. (A) MEC grown in db-CAMP-supplemented medium form a confluent squamous monolayer. ~156. (B) The same field as illustrated in A, 5 min after the addition of 10e5 M histamine in db-CAMP-supplemented growth medium. MEC maintained in db-CAMP do not express any morphological changes following histamine presentation. X156. (C and inset) Epithelial MEC grown to confluence in db-CAMP-supplemented medium. Cells were refed with db-CAMP-deficient medium 45 min

HISTAMINE

MODULATION

The peripheral vimentin filaments detach from the cell margin and contract toward the nucleus (Fig. 5B). The vimentin detachment is associated with the appearance of intercellular gaps in the formerly confluent epithelial cell sheet. Thereafter, the vimentin bundles composing the perinuclear reticular zone disassemble as the epithelial MEC acquire a transitional configuration (Figs. 5C and 5D). As the cells begin to reflatten, dense vimentin bundles reassemble into linear arrays, although portions of the radial skeleton may be retained (Fig. 5D). The encircling vimentin belt appears to be more resistant to depolymerization than the reticular matrix since it frequently persists after the loss of the vimentin reticulum. In the resulting plump bipolar transitional MEC, the vimentin matrix consists of loosely arranged linear arrays that conform to the irregular contours of the cell (Fig. 5D). Histamine-Calcium

Dynamics

The dynamics of calcium shifts in histamine-treated dermal MEC were analyzed with the calcium probe Fluo-3 and interactive laser cytometry. Unstimulated epithelial MEC, cultivated in db-CAMP-supplemented medium, possess relatively low free calcium levels approaching background (Figs. 7A and BA). Within a cell, free calcium in the central organelle-containing perinuclear zone, though low, was frequently 40-50% greater than that in the cells’ periphery (Fig. BA). When histamine is presented to db-CAMP-supplemented MEC, the cells exhibit a consistent low-level calcium response localized within the perinuclear and nuclear compartments (Figs. 7B and BA). Confocal microscopy reveals that free calcium in the nuclear compartment is distributed throughout the nucleoplasm and is not restricted to the perinuclear cistern. A dramatic difference in MEC free calcium distribution is observed when cultures are incubated in dbCAMP-deficient medium for 45 min prior to testing with histamine (Figs. 7C and BA). In the absence of exogenous db-CAMP, calcium levels become elevated throughout each of the MEC compartments. Within 1 s of histamine addition, such MEC exhibit a significant patterned and compartmentalized calcium response (Figs. 7D and 8). Levels of free Ca2+ within the reticular perinuclear compartment increase lOO%, while increases of only 50% above resting levels were seen in the cell periphery. Most strikingly, the MEC nuclei consis-

OF DERMAL

MEC

283

tently exhibited rapid, dramatic elevations in free calcium content, ranging from 200 to 400% over their initial concentration (Fig. 8). The nuclear calcium redistributed throughout the sponse is uniformly nucleoplasm as adjudged by confocal microscopy. Calcium levels generally peak within 10 s after exposure to histamine, and after 60 to 120 s the calcium levels drop sharply and plateau (Fig. BB). In about 10% of the studies, MEC exhibited two or three calcium oscillation peaks in the course of 5 min following histamine presentation (Fig. BB). MEC remain refractory to repeated additions of histamine through the duration of the observations. Interestingly, norepinephrine elicited an analogous, albeit lesser, shift in free calcium with the same compartmentalized distribution as that of histamine. It should be emphasized that MEC calcium responses to histamine and norepinephrine are independent of each other. Each agent will produce a response while the cells are in the presence of the other agent. The compartmentalized calcium response is characteristic of epithelial MEC and it is not observed in MEC expressing the mesenchymal phenotype. Transitional cells usually exhibit elevated levels of free Ca2+ prior to histamine stimulation. Such cells do not express significant changes in cytoplasmic Ca2+ upon addition of histamine. However, transitional cells with initially low levels of free Ca2+ express a Calcium response similar to that of epithelial MEC. In control cultures, analogously treated keratinocytes, melanocytes, and AG4431 fetal dermal fibroblasts did not show a calcium response upon exposure to histamine. Protein Kinase C Distribution Cytologic localization of protein kinase C was studied with fluorescein-conjugated monoclonal antibodies to the LYand p subunits of PKC. Epithelial MEC express a unique PKC distribution pattern which is primarily restricted to the vimentin-delimited nuclear and perinuclear compartments (Fig. 7E). PKC fluorescence within these zones reveals a distinct compartmentation similar to that observed for calcium. Quantification of antibody fluorescence indicates that the PKC concentration in the perinuclear zone is 3X higher and that in the nucleus is 6X higher than that in the attenuated peripheral zone. The spatial distribution and relative concentration of PKC coincide with those observed for cytoplasmic free calcium following histamine stimulation. PKC localiza-

before these fields were photographed. After these images were recorded, the cells were refed with 10m5M histamine in db-CAMP-free medium. X156. (D) Same fields illustrated in C and inset 1 min after histamine presentation. Junctional disruption and cell contraction result in the formation of intercellular gaps in the formerly confluent cell sheet. Many contracting cells maintain peripheral adhesive sites and consequently exhibit fine retraction spikes (arrowheads). Most contracted MEC reorganize as bipolar cells (arrows). Some cells completely roundup (double arrow) and frequently detach from the substrate. For orientation purposes, cells selected in C are identified with * in D (inset). In given fields, adjacent clusters of cells reveal differences in response to histamine. MEC cytoplasmic blebs resemble those on surfaces of contracting smooth muscle myocytes. X156.

284

LIPTON,

BENSCH,

AND

KARASEK

0.6

0.0

+

- -3H 4H -5H -6H

FIG. 2. Mitogenic response of dermal MEC to varying concentrations of histamine. Cell populations were determined by absorbance using a fluorometric hexosaminidase assay [28]. Absorbance readings reflect the mean of 8 to 12 assays in each category. The validity of the fluorometric assay was assessed by counting sample cell suspensions from each category in a hemocytometer. (+) MEC grown in standard db-CAMP-supplemented medium; (-) Cells cultivated in db-CAMP-free medium; (-3H, -4H, -5H, -6H) MEC cultures grown in db-CAMP-free medium containing 10m3,10m4,10m5,and 10m6M histamine, respectively.

tion and relative concentration remain essentially unchanged following histamine-stimulated cell contraction (Fig. 7F). It should be noted that PKC distribution is the same whether Triton X-100 or saponin is used to permeabilize the cells prior to staining with antibody.

1.4-

-r

1.2l.O-

0.80.60.40.20.0 +

-

H/+ H& '&3 4M C

m

D C&ID/H

FIG. 3. Histamine receptor expression in dermal MEC as determined by cell proliferation assays (see Fig. 2). Increases in mitotic activity observed with the H-l agonist 2-methylhistamine and the absence of an effect with H-2 agonist 4-methylhistamine suggest that H-l receptors mediate the MEC mitogenic response. Inhibition of the histamine proliferation response with H-l antagonist diphenhydramine and the absence of a response with the H-2 antagonist cimetidine support that conclusion. (+) MEC grown in standard db-CAMPsupplemented medium; (-) cells cultivated in db-CAMP-free medium); (H/+) db-CAMP-supplemented medium containing 10e5 M histamine; (H/k) db-CAMP-free medium containing 10e5 A4 histamine; (2M) db-CAMP-free medium supplemented with 10m5 M 2methylhistamine; (4M) db-CAMP-free medium supplemented with 10m5M 4-methylhistamine; (C) db-CAMP-free medium with 5 X 10e5 M cimetidine; (D) db-CAMP-free medium with 5 X 10m5M diphenhydramine; (C/H) cells fed with 5 X 10e5 M cimetidine with 10e5 M histamine subsequently added; (D/H) cells fed with 5 X 10e5 M diphenhydramine with 10e5 M histamine subsequently added.

FIG. 4. Laser cytometry reveals dermal MEC histamine receptors visualized with FITC-conjugated histamine. Confocal optics reveal that the receptors are localized to the apical (lumenal) surface overlying the nucleus and organelle-rich perinuclear zone. Receptor binding was inhibited by pretreating cells with excess unlabeled histamine or diphenhydramine. X270.

DISCUSSION

The main role of blood is to meet an organism’s nutritional and inflammatory demands. Microvessel endothelial cells differentially activate these blood functions through regulated phenotypic modulations [l]. In homeostatic vessels, transmural exchanges are mediated by epithelial MEC [3,5]. In acute inflammations, epithelial-mesenchymal transdifferentiations enable formerly confined immune-related cellular and humoral elements of the blood to cross the vessel walls selectively [5, lo]. In contrast to cell-mediated homeostatic exchanges, blood component distribution in inflammatory reactive microvessels is largely regulated by the basal lamina matrix. MEC epithelial-mesenchymal phenotypic transitions profoundly influence the function of microvessels [4,6, 7, 301. The expression of epithelial and mesenchymal phenotypes by cultivated dermal MEC is directly linked to levels of CAMP and free calcium [l, 25-271. Primary signals elevating CAMP have been shown to promote MEC epithelialization and enhance homeostatic functions [30-331. In contrast, vasoactive signals employing Ca2+ lead to intimal disruption and induction of inflammation-related mesenchymal traits [l, 18, 30, 32-341. Modulations of endothelial phenotype are largely brought about by alterations of the cytoskeletal matrix and cell junctions [l, 20, 331. Histamine, via Ca2+-mediated H-l receptors, causes a rapid elevation in free calcium levels in each of the three distinct cytological compartments that are formed by the vimentin cytoskel-

HISTAMINE

MODULATION

OF DERMAL

MEC

285

FIG. 5. Vimentin matrix expression in epithelial and histamine-modulated dermal MEC. (A) Epithelial MEC cultivated in db-CAMP-supplemented medium. Flattened, polygonal endothelial cells in a nearly confluent culture possess characteristic radially oriented and compartmentalized vimentin matrices. A differentiated epithelial MEC is illustrated at higher magnification in C. Matrix variations among cells in the field may reflect cell-cycle related changes in the cytoskeleton. The narrow spindle-shaped cells on the right margin are melanocytes. X264. (B) Extensive reorganization of vimentin cytoskeletons is observed in parallel cultures 1 min after addition of lo-’ M histamine in db-CAMP-free medium. Primary changes are the centripetal contraction of the peripheral zone vimentin filaments and the dissolution of reticular bundles comprising the perinuclear zone. The radially disposed vimentin matrices of polygonal epithelial MEC reorganize in parallel linear arrays that mark the emergence of a bipolar morphology (see cell marked with *). X264. (C) Characteristic vimentin matrix subdivides epithelial MEC into three compartments: (1) a thick sheath envelopes and compartmentizes the nucleus; (2) coarse reticular bundles radiating from the nuclear envelope and inserting in the circular belt delimit the organelle-rich perinuclear zone; and (3) an irregular feltwork of thinner vimentin bundles, extending from the belt to the cell margins, constitutes the peripheral zone. X464. (D) Following histamine presentation, the vimentin matrix in the former epithelial cell reorganizes to form dense linear bundles aligned with the cell’s anterior-posterior axis. Residual elements of the former perinuclear reticulum are still evident in the resulting transitional cell (arrows). X670.

eton of endothelial cells. Within seconds of histamine exposure, the radial actin-vimentin skeleton reorganizes into linear arrays that lead to the cell’s new bipolar morphology. The rapid onset of the MEC morphological

response (tl min) produced by inflammatory agents suggests that endothelial phenotypic modulation is initiated by post-translational alterations of existing gene products, rather than by the synthesis of new proteins.

286

LIPTON,

BENSCH,

AND

KARASEK

HISTAMINE

MODULATION

Post-translational modifications of vimentin have been shown to be differentially regulated by principal second messengers [35-381. Elevations in CAMP or Ca*+, with respective activation of either protein kinase A or C, appear to be major factors in both the remodeling of endothelial cells and the determination of microvessel function [30, 39-411. A causal relationship between PKC and MEC transdifferentiation is suggested by preliminary studies which reveal that H-7, a PKC inhibitor, prevents morphological reorganization of epithelial MEC [42]. The unique vimentin-associated compartmentation of Ca*+ and PKC in MEC activation has not been observed in other cultivated skin cells including fibroblasts, melanocytes, and keratinocytes. Fine structural studies indicate that the coarsely woven vimentin matrix cannot provide a physical barrier to limit the diffusion of calcium ions and enzymes [l]. Rather than defining a closed compartment, the vimentin matrix may serve as a physical substrate for the deployment of Ca*+ and PKC signals [43,44]. Regional localization of these regulatory agents may be related to vimentin binding of membranous calcium storage organelles (i.e., mitochondria and endoplasmic reticulum, Fig. 5) and cytoplasmic enzymes via intermediate filament-associated proteins (IFAP, [45-471). In addition to its role in structural support, vimentin may influence MEC phenotypic expression through its effect upon signal transduction. Polarized assemblies of intermediate filaments and IFAPs directly link plasmalemma receptors with specific organelles [45, 47, 481. Vimentin amino termini preferentially bind receptors at the plasmalemma, while the carboxy termini bind to lamin B of the nuclear envelope and possibly to other organelles [49-511. Vimentin post-translational alterations, subsequent to activation by Ca*+ or CAMP second messengers, may play an active role in signal transduction by converting receptor signals into enzyme activations and in the release of stored calcium from bound organelles [43,44,48]. The proposed role of vimentin as a physical substrate in supporting signal transduction would account for the compartmentalization of Ca*+ and PKC observed in activated MEC. Signal transduction in histamine-stimulated MEC is extremely rapid. Laser cytometer studies reveal that a uniform compartmentalized MEC calcium response oc-

OF DERMAL

MEC

287

curs within 100 ms of histamine presentation. Although an explanation of this finding is not yet available, it is provocative that organized linear assemblies of proteins (like those composing vimentin or collagen filaments), in addition to possessing high tensile strength, are endowed with semiconductor electrical properties (reviewed by [52]). The rapid distribution of the plasmalemma1 signal throughout the cell suggests that the inherent electrical conduction qualities of vimentin may participate in the process of signal transduction [53]. Vimentin-nuclear interactions have been implicated in genetic regulation. T. T. Puck and colleagues have provided evidence that CAMP-mediated alterations of vimentin may regulate gene activation through the selective exposure of certain genes to transcription elements and the sequestration of others [54-561. Similarly, Ca*+-activated post-translational modifications of vimentin subunits have also been shown to affect sitespecific DNA-vimentin binding characteristics differentially [45]. Collectively, the compartmentalized nuclear calcium response, the high nuclear PKC content, and the simultaneous alterations in the vimentin perinuclear sheath implicate vimentin with a major role in regulating gene expression of MEC in inflammatory tissues. A similar role for PKC-mediated vimentin posttranslation in regulating signal transduction in human lymphocytes has been reported by Liebowitz et al. [43]. Extending these studies, Guy and Gordon [44] propose a cell control system composed of a network of several “activation circuits” which, individually or in combination, may be stimulated to produce a common result. They suggest that at some point in each signal cascade a common signal integrator controls a crucial event such as expression of a specific gene. Similarly, MEC transdifferentiation may be elicited by a number of primary signals, including (but not limited to) norepinephrine, bradykinin, substance P, and phorbol esters. Although MEC possess a variety of different receptor-mediated activation circuits, the phenotypic consequence of the resulting signal cascade is ultimately dependent upon which of the two “signal integrators,” CAMP or Ca*+, is used [22, 30-331. The antagonism of CAMP and Ca2+ second messengers on endothelial expression provides insight into the basic duality of the receptors for the major vasoactive

FIG. 6. Fine structure of intermediate filament (IF) cytoskeleton association with the nucleus and membranous organelles in epithelial MEC. (N) nucleus; (M) mitochondria; (W) Weibel-Palade bodies; (E) endoplasmic reticulum; (V) multivesicular body. (A) Tangential section grazing nuclear envelope and revealing nuclear pores. Bundles of IFS closely adherent to the nuclear envelope compose the vimentin nuclear sheath (see Fig. 4). IFS, which appear to terminate on or near the nuclear pores, radiate outward through the cytoplasm making intimate contact with membranous organelles. (inset) Cross section through the nuclear envelope shows filamentous densities adherent to cytoplasmic face of a nuclear pore (arrow). Cytoskeletal filaments enmesh adjacent mitochondria. ~35,000; inset, X53,200. (B) An example of a coarse IF bundle constituting part of the perinuclear reticulum. Mitochondria are frequently aligned and enmeshed within these IF bundles. x15,700. (C) Bundles of IFS make an abrupt turn and appear to terminate on membrane of endoplasmic reticulum. X55,000. (D) Cross section through MEC perinuclear zone. Dense reticular bundles of IFS course through the cytoplasm enmeshing mitochondria, cisterns of endoplasmic reticulum, and a multivesicular body. ~50,000.

288

LIPTON,

BENSCH,

AND

KARASEK

HISTAMINE

MODULATION

OF DERMAL

MEC

289

lated, Rasmussen did not have an example of a cell agents, including adrenalin, histamine, and acetylchowhose primary activity was mediated by CAMP and anline. Despite the wide range of vasoactive agents, microtagonized by Ca*+. The results of this study fill that gap. vessel endothelial cell expression is restricted to two The primary behavior of MEC, homeostatic regulation, primary functional/structural configurations. Elevated is mediated by CAMP and inhibited by Ca2+. These studintracellular Cazf levels due to stimulation of cr, H-l, ies also reveal that the presence of exogenous CAMP is and muscarinic receptors lead to expression of an “ineffective in inhibiting a calcium response and subseflammatory” MEC phenotype [5, 57, 581. In contrast, of dermal MEC by receptors stimulating increases in CAMP levels (e.g., /3, quent morphological reorganization H-2, or nicotinic) produce intimal epithelialization [32, histamine. Elevated CAMP has also been shown to inhibit inflammatory-related calcium responses signifi33,591. Quantitative or qualitative differences in recepcantly in macrovessel endothelial cells [59]. tor populations employing CAMP and Ca2+ as second An understanding of the opposing activities of CAMP messengers may account for regional specializations of and Ca2’ second messengers in mediating the structural microvessels and tissue specific MEC heterogeneity. The antagonistic relationship between Ca2+ and duality of MEC may be helpful in elucidating the pathoCAMP in regulating cell phenotype is not restricted to genetic mechanisms underlying specific vascular abnormalities. In diabetes, for example, microvessel endothemicrovessel endothelial cells. A similar dual CAMPha1 cells express an increased calcium-dependent proCa*+ modulation pathway influencing cytoskeletal syntein kinase C activity [64]. Such increased enzyme thesis and assembly has been found to occur in mouse activity would be expected to induce MEC mesenchyosteoblastic cells [60]. The differential effects of CAMPmalization and lead to chronic vascular leakage. T. M. dependent and independent pathways on epithelialmesenchymal expression in a variety of cell types and Hollis has shown that vascular stress, perhaps mediated cell lines has recently been reviewed by Dumont et al. by high glucose levels, stimulates endogenous histidine [61]. Particularly interesting are the described relationdecarboxylase activity in endothelial cells [65-671. ships between CAMP and over-secreting epithelial ade- These studies reveal that an elevation in plasma histanomas, and the influence of Ca*+ levels on undifferenmine, rather than glucose, may be responsible for the tiated fibroblast-like tumors. increased PKC activity observed in diabetes. Blocking The duality and interrelationship of CAMP and Ca2+ the histamine-calcium pathway through infusion of as universal intracellular second messenger systems H-l antagonists results in an inhibition of MEC mesenhave been addressed by Rasmussen [62,63]. In coining chymalization and associatedvascular leakage in experithe term “synarchic regulation” to describe the interacmental diabetes [68,69]. The effect is so profound that antihistamine infusion into streptozotocin-induced diation of second messengers and their roles in regulating cell processes, Rasmussen identified five variations: co- betic rats reduces retinal vascular leakage to such an ordinate, sequential, redundant, hierarchical, and antagextent that it may prevent diabetic retinopathy [68,69]. onistic. In the last mentioned class of interaction, examIn preliminary clinical trials treating human diabetics ples in which CAMP serves as an inhibitory influence on with antihistamines, the Hollis group has demonstrated cells whose primary functions rely on Ca*+-mediated the efficacy of these agents in lessening vascular leakage signals were cited. Although its existence was postuand stemming retinal degeneration [70].

FIG. 7. Fluorescence pseudo-images of MEC produced by laser cytometry. The color values scale (lower right corner) refers to relative fluorescence and applies to all micrographs in the plate. (A) Epithelial MEC grown in db-CAMP-supplemented medium. The peripheral margins of the cells in this confluent epithelium are not distinct. However, elevated levels of free calcium in the perinuclear zone serve to distinguish individual cells. X380. (B) Same field of cells illustrated in A, 10 s after addition of 10e5 M histamine. Although calcium fluorescence is relatively low in db-CAMP-supplemented MEC, the concentration of free calcium in the nucleus and perinuclear compartment doubles following histamine presentation (see Fig. 8A). Free-calcium in the MEC peripheral compartment is not affected by histamine. x380. (C) Epithelial MEC in cultures preincubated in db-CAMP-free medium for 45 min prior to imaging calcium levels. In one selected cell the nuclear, perinuclear, and peripheral zones have been traced and digitized for quantitating calcium fluxes (see Fig. 8B). Cytoplasmic free calcium levels are significantly higher in MEC following incubation in db-CAMP-free medium (see Fig. 8A). Free calcium in the nucleus and organelle-rich perinuclear zone is characteristically greater than that in the attenuated cell periphery. X340. (D) Histamine-induced calcium response in same field illustrated in C. Ten seconds after histamine presentation, significantly elevated Ca *+ levels exhibit a nonuniform distribution. Histamine produces calcium increases of 50% in the cell periphery, 100% in the perinuclear zone, and 200-400% in the cell nuclei (see Fig. 8). Confocal microscopy reveals that the free calcium is uniformly distributed throughout the nucleoplasm and not restricted to the perinuclear cistern. X340. (E) Protein kinase C (PKC!) distribution in epithelial MEC. PKC was localized with FITC-conjugated antibodies against the (Yand S isoforms. PKC distribution is primarily restricted to the vimentin-delimited nuclear and perinuclear zones. The distinctive compartmentation of PKC is similar to that observed for Ca2+ in histamine-treated MEC (see Fig. 7D). Based on relative fluorescence, PKC concentration in the perinuclear zone is 3X higher, while nuclear PKC is approximately 6X higher than that in the peripheral zone. X380. (F) PKC distribution in MEC following histamine activation. MEC preincubated for 45 min in db-CAMP-deficient medium and subsequently exposed for 5 min to 10m5M histamine express a transitional phenotype. In such cells, the PKC maintains the same nuclear and perinuclear distribution as in unstimulated MEC (see E). X270.

290

LIPTON,

+N

0

55

+PN

+P

110

165

Time

-N

-PN

220

215

BENSCH,

-P

330

(set)

FIG. 8. Calcium dynamics in histamine-treated MEC. (A) Quantitation of histamine-induced calcium flux as determined by changes in relative fluorescence. Graph compares relative fluorescence (mean + SE) in traced and digitized nuclear, perinuclear, and peripheral compartments of 24 cells, before and after histamine presentation. MEC maintained in CAMP exhibit a significantly depressed calcium response when exposed to histamine. The graded calcium response in CAMP-deficient cells is statistically significant (S < 0.000) among all three compartments. Histamine induced significant increases in calcium in each of the three defined MEC compartments: nucleus = 232 f 9.4% (mean + SE), perinuclear compartment = 102 + 5.6%, and peripheral compartment = 51 f 3.0%. (N) nuclear compartment; (PN) perinuclear compartment; (P) peripheral compartment; (El) To; (m) 10 s after histamine presentation; (+) cells maintained in dbCAMP-supplemented medium; (-) cells preincubated in db-CAMPdeficient medium 45 min prior to assay. (B) Relative changes in cytoplasmic free-calcium within three cells numbered in Fig. 7D. The nuclear, perinuclear, and peripheral zones were traced and digitized (see example in Fig. 7C). Resting levels of calcium were determined at T0 and histamine was added at 45 s, 10 s prior to the second scan. Subsequent scans were made at 55-s intervals. Relative changes in free calcium in each of the cell’s three compartments were quantitized over time using the ACAS kinetics program. Histamine-induced calcium oscillations are observed in Cell 1. (- - -) Cell 1; (- --) Cell 2; (-) Cell 3; (N) nuclear compartment; (PN) perinuclear compartment; (P) peripheral compartment; (arrow) time of histamine addition.

In conclusion, studies on MEC behavior and cytology support the following model of functional regulation of microvessel expression: Differentiated dermal MEC are genetically programmed to express both epithelial and mesenchymal phenotypes. The homeostatic epithelial configuration is selected through primary signals em-

AND

KARASEK

ploying CAMP as a second messenger. Cyclic AMP-activated post-translational enzymes modify vimentin matrix and epithelial junctional components generating a radial epithelial cytoskeleton. In the nucleus, CAMPmodulated vimentin proteins may regulate DNA binding patterns that support grouped epithelial functions but repress mesenchymal programs. Calcium-activated post-translational alterations of cytoskeletal components, following trauma or inflammation, lead to junctional dissolution and vimentin reorganization. While MEC structural modulations are immediate, a delayed secondary response, possibly related to altered vimentin-DNA associations, leads to activation of mesenchymal genes and repression of epithelial programs. We propose that the fine-tuning of basic epithelial or mesenchymal phenotypes, further specifying appropriate endothelial functions, is controlled by environmental signals (e.g., hormones, cytokines, and extracellular matrix components). Chronic elevated histamine due to inflammatory stimuli or erratic vascular shear stresses stimulates MEC proliferation. Modulation of the resulting MEC progeny by environmental factors may lead to either epithelial hemangiomas and endotheliomas or to mesenchymai hyperplasias such as Kaposi’s sarcoma, intimal sclerotic plaques, granulomas, or fibroplasias. The results of this study provide insight and a testable model for the molecular mechanisms underlying the morphological and functional expressions of microvessel endothelial cells. Through an understanding of the role of second messengers and their influence on vimentin cytoskeletal components it may be possible to regulate the signal cascade that controls the activation of homeostatic and inflammatory expressions in health and disease. The authors thank Irma Daehne, Mary Jane Eaton, and Mary Kovats for their excellent technical assistance. We also thank Meridian Instruments and Barbara J. Laughter for technical support of the laser cytometer assays.

REFERENCES 1.

Lipton,

B. H., Bensch, K. G., and Karasek,

M. A. (1991) Differ-

entiation 46, 117-133. 2. 3. 4. 5. 6. 7.

Casley-Smith, J. R. (1980) Adu. Microcirc. 9, l-44. Gerritsen, M. E. (1987) Biochem. Pharmacol. 36, 2701-2711. Simionescu, N. (1983) Physiol. Reu. 63, 1536-1579. Crone, C. (1986) Fed. Proc. 45, 77-83. Pober, J. S. (1988) Am. J. Puthol. 133,426-433. Mantovani, A., and Dejana, E. (1989) Zmmunol. Today 10,370-

375. 8. Majno, G., and Palade, G. E. (1961) J. Biophys. Biochem. Cytol. 11,571-603. 9. Gimbrone, M. A. (1986) in Vascular Endothelium in Hemostasis and Thrombosis Edinburgh.

(Gimbrone,

M. A., Ed.), Churchill

Livingstone,

HISTAMINE 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

31. 32. 33.

34.

35. 36. 37. 38.

Movat,

H. 2. (1987) Can. J. Physiol. Pharmacol.

65,451-457.

Robbins, S. L., Cotran, R. S., and Kumar, V. (1984) Pathological Basis of Disease, 3rd ed., Chapt. 2, Saunders, Philadelphia. Bardadin, K. A., and Desmet, V. J. (1985) Histopathol. 9, 171181. Beranek, J. T. (1989) Med. Hypotheses 28, 271-273. Kocher, O., and Madri, J. A. (1989) In Vitro Cell Deu. Biol. 25, 424-434. Schmid, V. (1988) Cell Differ. Dev. 22, 173-182. Beresford, W. A. (1990) Cell Differ. Deu. 29,81-93. Braverman, I. M., and Keh-Yen, A. (1986) J. Invest. Dermutol. 86,577-581. Northover, A. M., andNorthover, B. J. (1985) in The Pharmacology of Inflammation (Bonta, I. L., Bray, M. A., and Parnham, M. J., Eds.), Vol. 5, pp. 235-247, Elsevier, Amsterdam. Owen, D. A. A., Pipkin, M. A., and Woodward, D. F. (1984) Agents Actions 14, 39-42. Rotrosen, D., and Gallin, J. I. (1986) J. Cell Biol. 103, 23792387. Hallam, T. J., Jacob, R., and Merritt, J. E. (1988) Biochem. J 255,179-184. Adams, D. J., Barakeh, J., Laskey, R., and Van Breemen, C. (1989) FASEB J. 3, 2389-2400. Neylon, C. B., and Irvine, R. F. (1991) J. Biol. Chem. 266,42514256. Marks, R. M., Roche, W. R., Czerniecki, M., Penny, R., and Nelson, D. S. (1986) Lab. Inuest. 55, 289-294. Karasek, M. A. (1989) J. Znuest. Dermatol. 93, 33S-38s. Bensch, K. G., Davison, P. M., and Karasek, M. A. (1983) J. Ultrastruct. Res. 82, 76-89. Tuder, R. M., Karasek, M. A., and Bensch, K. G. (1990) J. Cell. Physiol. 142, 272-283. Landegren, U. (1984) J. Zmmunol. Methods 67, 379-388. Merritt, J. E., McCarthy, S. A., Davies, P. A., and Moores, K. E. (1990) Biochem. J. 269.513-519. Smirnov, V. N., Voyno-Yasenetskaya, T. A., Antonov, A. S., Lukashev, M. E., Shirinsky, V. P., Tertov, V. V., and Tkachuk, V. A. (1990) Ann. N.Y. Acad. Sci. 598, 167-181. Stelzner, T. J., We& J. V., and O’Brein, R. F. (1989) J. Cell. Physiol. 139,157-166. Oliver, J. A. (1990) J. Cell. Physiol. 145, 536-542. Yamada, Y., Furumichi, T., Furui, H., Yokoi, T., Ito, T., Yamauchi, K., Yokota, M., Hayashi, H., and Saito, H. (1990) Arteriosclerosis 10,410-420. Hallam, T. J., Merritt, J. E., Rink, T. J., and Jacob, R. (1989) in Vascular Endothelium: Receptors and Transduction Mechanisms (Catravas, J. D., Gillis, C. N., and Ryan, U. S. Eds.), Vol. 175, pp. 165-172, Plenum, New York. Evans, R. M. (1988) FEBS Lett. 234,73-78. Huang, C., Devanney, J. F., and Kennedy, S. P. (1988) Biochem. Biophys. Res. Commun. 150, 1006-1011. Ando, S., Tanabe, K., Gonda, Y., Sato, C., and Inagaki, M. (1989) Biochemistry 28, 2974-2979. Geisler, N., Hatzfeld, M., and Weber, K. (1989) Eur. J. Biochem.

183,441-447. 39.

Mackie, K., Lai, Y., Nairn, A. C., Greengard, P., Pitt, B. R., and Lazo, J. S. (1986) J. Cell. Physiol. 128, 367-374.

Received July 7,199l Revised version received October 21, 1991

OF DERMAL

MODULATION 40. 41. 42. 43. 44. 45. 46. 47. 48. 49 50. 51. 52. 53.

54 55. 56. 57. 58.

MEC

291

Kazlauskas, A., and DiCorleto, P. E. (1987) J. Cell. Physiol. 130, 228-244. Lynch, J. J., Ferro, T. J., Blumenstock, F. A., Brockenauer, A. M., and Malik, A. B. (1990) J. Clin. Znuest. 85, 1991-1998. Lee, J., and Karasek, M. (1990) Clin. Res. 38, 224A. Liebowitz, D., Kopan, R., Fuchs, E., Sample, J., and Kieff, E. (1987) Mol. Cell. Biol. 7, 2299-2308. Guy, G. R., and Gordon J. (1989) Znt. J. Cancer 43,703-708. Traub, P. (1985) Ann. N.Y. Acad. Sci. 455, 68-78. Wang, E. (1985) Ann. N.Y. Acad. Sci. 455,32-56. Markl, J. (1991) J. Cell Sci. 98, 261-264. Goldman, R., Goldman, A., Green, K., Jones, J., Lieska, N., and Yang, H. (1985) Ann. N.Y. Acud. Sci. 455,1-17. Georgatos, S. D., Weaver, D. C., and Marchesi, V. T. (1985) J. Cell Biol. 100, 1962-1967. Georgatos, S. D., and Blobel, G. (1987) J. Cell Biol. 105, 117125. Steinert, P. M., and Liem, R. K. H. (1990) Cell 60, 521-523. Szent-Gyorgyi, A. (1960) An Introduction to a Subcellular Biology, pp. 50-53, Academic Press, New York. Adey, W. R. (1988) in Electromagnetic Fields and Neurobehavioral Function (O’Connor, M. E., and Lovely, R. H., Eds.), Vol. 257, pp. 81-106, A. R. Liss, New York. Puck, T. T. (1987) Somatic Cell Mol. Genet. 13,451-45'7. Ashall, F., Sullivan, N., and Puck, T. T. (1988) Proc. Nutl. Acad. Sci. USA 85, 3908-3912. Chan, D., Goate, A., and Puck, T. T. (1989) Proc. Natl. Acud. Sci. USA 86,2747-2751. Northover, A. M. (1988) Agents Actions 24,351-355. Sim, M. K., and Manjeet, S. (1990) Eur. J. Phurmacol. 189,

399-404. 59. 60. 6l ’ 62. 63. 64 f 65. 66 ’ 67. 68.

69. 70.

Carson, M. R., Shasby, S. S., and Shasby, D. M. (1989) Am. J. Physiol. 257, L259-264. Lomri, A., and Marie, P. J. (1990) Biochim. Biophys. Acta 1052, 179-186. Dumont, J. E., Jauriaux, J.-C., and Roger, P. P. (1989) Trends Biochem. Sci. 14,67-71. Rasmussen, H. (1982) Bull. Schweiz. Akad. Med. Wiss. 82,131147. Rasmussen, H. (1982) in Hormone Receptors (Kohn, L. D., Ed.), pp. 175-197, Wiley, New York. Lee, T. S., Sattsman, K. H., Ohashi, H., and King, G. L. (1989) Proc. Natl. Acad. Sci. USA 86,5141-5145. Hollis, T. M., Gallik, S. G., Orlidge, A., and Yost, J. C. (1983) Arteriosclerosis 3, 599-606. Carroll, W. J., Hollis, T. M., and Gardner, T. W. (1988) Inuest. Ophthalmol. Visual Sci. 29,1201-1204. Skarlatos, S. I., and Holiis, T. M. (1987) Atherosclerosis 64,5561. Hollis, T. M., Gardner, T. W., Vergis, G. J., Kirbo, B. J., Butler, C., Dull, R. O., Campos, M. J., andEnea, N. A. (1988) J. Diabetic Complications 2, 47-49. Enea, N. A., Hollis, T. M., Kern, J. A. G., and Gardner, T. W. (1989) Arch. Ophthulmol. 107,270-274. Gardner, T. W., Eller, A. W., Friberg, T. K., D’Antonio, J. A., Fuderich, M. E., and Ostraska, P. (1991) Inuest. Ophthalmol. Visual Sci. 32. 1289.