ARTICLE
Ambulatory venous hypertension underlies the pathogenesis of chronic
venous insufficiency (CVI) and ulceration [1], but the pathophysiological
steps leading from venous hypertension to ulceration are still ill-defined.
Recent CVI research detected changes in the microcirculation of the skin
accompanied by an altered expression of growth factors, such as PDGF-alpha
and beta or VEGF [2-5]. It has been hypothesized that macromolecules extravasated
from the vasculature bind growth factors and matrix proteins [6]. Growth
factors are signalling peptides that act through specific cell surface
receptors and cause paracrine or autocrine stimulation of cell proliferation
and migration [7]. Efforts have been made to administer by topical treatment,
single or a mixture of exogenous growth factors to chronic wounds or leg
ulcers [8, 9].
Epidermal growth factor (EGF) is a 6 kDa polypeptide, which shares a
common receptor with transforming growth factor-alpha, termed the EGF
receptor (EGFR). EGFRs, transmembrane proteins with a molecular weight
of 170 kDa, are present in all non-hematopoetic cells. EGF induces proliferation
of epidermal basal cells, endothelial cells, fibroblasts, and muscle cells
[7, 10, 11]. It stimulates cell hyperplasia and increases activity of
epidermal enzymes and the synthesis of glycosaminoglycans by human fibroblasts,
but may inhibit collagen deposition [11, 12].
Fibroblast growth factor (FGF) exists in at least 8 closely related
forms: the most widely studied, basic FGF and acidic FGF sharing 55% homology.
Basic FGF is a single chain polypeptide with a molecular weight of 17
kDa [7]. Cell-associated heparan sulfate seems to coordinate the interaction
between bFGF and its 170 kDa receptor [13]. Fibroblasts, endothelial cells,
and keratinocytes produce bFGF in vitro. FGF affects morphology,
proliferation, and differentiation of mesoderm-derived cells, including
endothelial cells, and of cells of ectodermal origin, including melanocytes,
glial cells, and neuroblasts. FGF modulates the pattern of collagen expression
and fibronectin synthesis [10].
Transforming growth factor-beta (TGF-ß) is a 25 kDa homodimer
and can be found in almost all tissues [7, 14]. TGF-ß has three
distinct highly specific receptors with molecular weights of 53, 73, and
130 kDa that are present in all cells. TGF-ß regulates cell proliferation
and connective tissue synthesis stimulating mitosis of mesenchymal cells
such as fibroblasts, and inhibiting the growth of other cells such as
keratinocytes, vascular endothelial cells, and T- and B-lymphocytes [10,
14]. TGF-ß increases extracellular matrix deposition of collagen,
proteoglycans, and fibronectin [15].
The role of growth factors in pathological conditions of the skin has
been best examined in the healing of acute and chronic wounds [6, 7],
e.g. TGF-ß3, a new isoform of the TGF-ß superfamily
is presumed to play an important role in wound repair [16]. Recently,
it has been shown that the reduced growth of dermal fibroblasts from chronic
venous ulcers can be stimulated by growth factors, such as bFGF and EGF
[17]. In the present study, we have used immunofluorescence and immunoperoxidase
techniques to evaluate the pathogenic relevance of EGFR-, bFGF-, and TGF-ß3-expression
pattern in progressing stages of CVI.
Patients and methods
Patients
The study group included 30 patients (22 women, 8 men) with different
stages of CVI, and an average age of 65 years (44-83 years). Patients
were divided into 5 different groups according to progressing skin changes
ascribed to venous disease [18] : group 1 telangiectases and reticular
veins, group 2 venous eczema, group 3 pigmentation, group
4 lipodermatosclerosis and group 5 leg ulcer. There were
six patients in each group. Clinical diagnosis was confirmed by continuous-wave
(CW) Doppler and photoplethysmography. Exclusion criteria included a brachio-pedal-index
below 0.9, indicating arteriosclerosis. As a control group, six age- and
sex-matched patients were taken, who had no signs of chronic venous insufficiency
on continous-wave (CW) Doppler and photoplethysmography. After application
of local anaesthesia (1% Scandicain), following written, informed consent,
6 mm punch biopsies were taken from those areas of the lower leg showing
clinical signs of CVI.
Frozen sections
Biopsies were immediately snap frozen in liquid nitrogen and stored
at 80° C. To obtain 5 µm cryostat sections, they were
embedded in Tissue Freezing Medium (Jung, Nussloch, Germany). The sections
were mounted onto gelatin-coated glass slides and dried (12 h x 20°
C). Afterwards they were stored at 20° C.
Antibodies
Monoclonal antibody mouse anti-human epidermal growth factor receptor
(DAKO, Glostrup, Denmark) and polyclonal antibodies rabbit anti-human
transforming growth factor ß3 (III) (IC Chemikalien, Ismaning, Germany),
and rabbit anti-human basic fibroblast growth factor (a kind gift of Pr.
Dr. C. Grothe, Department of Anatomy, University of Freiburg, Germany),
were used as primary antibodies. Primary antibody mouse anti-human IgG
(Dako, Hamburg, Germany) was used as inappropriate antibody.
Second layer antibodies were Cy-3 goat anti-mouse-IgG (Dianova, Hamburg,
Germany) and link antibodies goat anti-mouse-IgG (Sigma, St. Louis, MO)
and goat anti-rabbit-IgG (Sigma, St. Louis, MO). Mouse peroxidase anti-peroxidase
(Sigma, St. Louis, MO) and rabbit peroxidase anti-peroxidase (Sigma, St.
Louis, MO) were used as PAP antibodies.
Indirect immunofluorescence
Cryostat sections were dried (20 min x 20° C) and fixed in acetone
(Merck, Darmstadt, Germany; 10 min x 20° C). The alternative fixation
procedure was the application of paraformaldehyde (Merck, Darmstadt, Germany,
4% w/v; 10 min x 20° C) and permeabilisation by Triton X-100 (Merck,
Darmstadt, Germany, 0,5% v/v; 10 min x 20° C). The fixed tissues
were washed in phosphate buffered saline (PBS, Gibco BRL, Paisley, Scotland,
pH 7.2; 10 min x 20° C). After preincubation with bovine serum albumin
(BSA, Serva, Heidelberg, Germany, 3% w/v; 20 min x 20° C) to reduce
background staining, the primary antibody was added (3 hrs x 37°
C). The sections were washed in PBS (10 min x 20° C) and the secondary
antibody, that had been centrifuged earlier (3 min x 3,000 g), was applied
(2 hrs x 37° C). After repeated washings in PBS (10 min x 20°
C), sections were mounted in Tris-buffered Moviol (Hoechst, pH 8.6) and
examined under epifluorescence illumination on a Zeiss Axioskop microscope
with a 20x Zeiss objective, using a BP 510-560, FT 580, LP 590 filter.
Controls, where either the primary antibody was omitted or inappropriate
primary antibodies were used, showed no cross reaction between the labelled
reagent. Sections were photographed using Ektachrome Elite (400ASA) film
(Eastman-Kodak).
PAP immunoperoxidase
Cryosections were fixed in paraformaldehyde (Merck, Darmstadt, Germany,
4% w/v; 10 min x 20° C), rinsed in PBS (pH 7.2; 10 min x 20°
C) followed by preincubation with dilution buffer (Triton X-100, Merck,
Darmstadt, Germany, 0,5% v/v + milk powder, 3% w/v in PBS; 30 min x 20°
C). After washing in PBS (10 min x 20° C), sections were incubated
with primary antibodies in dilution buffer (2 hrs x 37° C) and rinsed
in PBS (10 min x 20° C). Then the secondary antibodies in dilution
buffer were applied (1 h x 37° C). Before and after incubation with
the PAP antibodies in dilution buffer (1 h x 37° C), sections were
washed in PBS (10 min x 20° C). Diaminobenzidine tetrahydrochloride
(DAB, DAKO, Glostrup, Denmark; 0,14% w/v; 10-50 min x 20° C) was
used as chromogen. Sections were counterstained with haematoxylin (Fluka,
Buchs, Switzerland; 15 s x 20° C) and mounted in Kaiser`s glycerol
gelatin (Merck, Darmstadt, Germany). Application of dilution buffer or
inappropriate primary antibodies replacing primary antibodies were used
as controls. Results were analysed on a Zeiss Axioskop microscope with
a 20x Zeiss objective and photographed with Ektachrome 64T film (Eastman-Kodak).
Semiquantitative evaluation of growth factor expression
Growth factor expression in the skin was scored by two independent observers
in a double-blind manner. Staining of the epidermis, the capillaries,
and the connective tissue cells were judged from 0 to +++ using the criteria
described in the legend of Table
I.
Results
The results of EGFR-, bFGF-, and TGF-ß 3-expression pattern in
normal skin and progressing stages of CVI are summarized in Table
I. There were six patients in each group (total number of patients,
n = 36). In normal skin of the control patients, EGFR was expressed mainly
in basal epidermal cell layers with a decreased staining pattern towards
the surface, complete absence was noted in the stratum corneum (Fig.
1A). Moreover, EGFR expression was seen in few connective tissue
cells, perivascular cells and capillary endothelial cells. A similar expression
pattern was seen in skin with telangiectases and reticular veins. In contrast,
the epidermis with venous eczema showed a homogeneous EGFR expression,
including an intensive staining of the suprabasal layers (Fig.
1B). In addition, EGFR-positive stromal cells increased at this
stage of CVI. EGFR expression in skin with pigmentation decreased and
did not significantly differ from normal skin or that with telangiectases
and reticular vein skin. In sections with lipodermatosclerosis expression
of EGFR was visible in all epidermal layers, however, more predominantly
in the basal cell layers. Furthermore, the number of stained dermal endothelial
cells once again increased (Fig.
1C). Connective tissue cells and capillaries in the dermis of
leg ulcer specimens strongly expressed EGFR. In the epidermis, positive
staining was limited to the basal cell layer.
Normal epidermis and epidermis with telangiectases and reticular veins
showed a weak bFGF expression. Within the epidermis, only the basal cell
layer was stained positive. Basic FGF showed a faint expression in endothelial
and stromal cells like fibroblasts (Fig.
1A'). In contrast, suprabasal cell layers demonstrated an increased
expression of bFGF in the epidermis with venous eczema. The amount of
stromal cells marked by the bFGF antibody was raised in the papillary
layer of the corium (Fig. 1B').
The bFGF staining pattern of the skin with pigmentation was comparable
to that of normal skin. However, in specimens of lipodermatosclerotic
skin, basal and suprabasal cell layers were moderately stained, whereas
bFGF was expressed strongly by basal epidermal cells in leg ulcer skin.
Basic FGF expression of endothelial cells and stromal cells was increased
again in lipodermatosclerotic skin progressing to leg ulcer, where it
reached its highest expression (Fig.
1C').
TGF-ß3 antibody stained endothelial cells of capillaries and connective
tissue cells, mainly located in the papillary layer of corium, weakly
in normal skin and skin with telangiectases and reticular veins (Fig.
1A''). The immunoreactivity on leukocytes, and to a lesser extent,
the number of TGF-ß3 positive vascular endothelial cells increased
in venous eczema skin (Fig. 1B'').
Resembling the bFGF and EGFR expression pattern in the dermis of pigmentation
skin, TGF-ß3 staining exhibited a comparable expression in specimens
of pigmentation skin similar to that of normal skin. In lipodermatosclerotic
skin, TGF-ß3 antibody marked as many vessels and subepidermal cells
as in venous eczema. Peak levels of TGF-ß3 staining were seen in
endothelial cells and connective tissue cells of the leg ulcer base (Fig.
1C''). A weak TGF-ß3 expression of epidermal basal cells
was visible within all stages of CVI.
Discussion
In the present study, we have shown by immunohistochemistry that EGFR,
bFGF, and TGF-ß3 expression is markedly increased in skin biopsies
of venous eczema and leg ulcers, and to a lesser extent in specimens of
lipodermatosclerosis. Oxidative stress in CVI, caused by ambulatory venous
hypertension in the lower legs, which results in changes in the microvascular
architecture may affect the cellular hydration state [1, 19]. Epithelial
cell swelling, as seen with venous eczema and lipodermatosclerosis, stimulates
protein synthesis, and changes in membrane tension, cellular ion concentrations
and cytoskeletal architecture [20]. Therefore, cell swelling, that can
be caused by oxidative stress, hormones, and cytokines [21], may be an
important step towards changes of EGFR, bFGF and TGF-ß3 expression
in skin with CVI.
EGF receptors are present in increased numbers in the hyperproliferative
epidermis of proliferative skin diseases such as active psoriasis [11],
wound healing [22] and benign acantotic dermatoses [23]. Intense localization
of EGF receptors in the epidermis of venous eczema and lipodermatosclerosis
suggests that the requisite receptors for EGF and its ligands are expressed
in a keratinocyte population with an amplified proliferation rate in these
stages of CVI. Furthermore, EGF is a fundamental regulator of epithelial
differentiation and elicits distinctive changes in cytokeratin expression
[24]. Regarding our previous studies, we described alterations of cytokeratin
10 and 14 expression in the epidermis of venous eczema and lipodermatosclerosis
[25]. The lack of EGF receptor expression in the wound edges of leg ulcers
might result in diminished proliferation and motility of keratinocytes,
which might be responsible for the poor healing properties of chronic
leg ulcers. We observed an elevated EGFR expression in the dermis of venous
eczema, lipodermatosclerosis and leg ulcer, where inflammation or connective
tissue remodulation occurs. Hyperplasia and hypertrophy of mesenchymal
cells including fibroblasts, endothelial cells and smooth muscle cells
can be stimulated by EGF. Besides, EGF influences the synthesis of glycosaminoglycans
and collagens by fibroblasts [24].
Basic FGF has been shown to be nearly as effective as EGF in stimulating
keratinocyte growth [26, 27]. In normal human epidermis, bFGF immunoreactivities
are observed in the first 2-3 layers of basal and suprabasal keratinocytes,
while in the case of psoriasis, positive immunoreactivities are also seen
in the upper suprabasal layers [26]. Our results show that suprabasal
cell layers of venous eczema and lipodermatosclerotic epidermis show a
bFGF positive staining, indicating that increased keratinocyte proliferation
appears in these stages of CVI. However, in specimens of leg ulcer edges,
only the basal epidermal layer expresses basic FGF, suggesting that basic
FGF synthesis in the epidermis of ulcer edges is deficient compared to
normal skin. Basic FGF binds to heparan sulfates in the extracellular
matrix [13], it stimulates collagenase synthesis matrix deposition [26],
and seems to be involved in organizing connective tissue sclerosis. These
findings are in accordance with the observed expression peaks in the dermis
of lipodermatosclerosis and chronic leg ulcer wounds, which are clinically
characterized by an increasing sclerotic process.
Beside the mitogen stimulation of fibroblast proliferation, differentiation
and modulation of the mononuclear cell infiltrate of the skin [14], TGF-ß
exhibits a potent antiregulative effect on epithelial cells [27], vascular
endothelial cells, and lymphocytes, and inhibits EGF-dependent endothelial
cell proliferation [8, 12]. Strongly increased TGF-ß3 expression
on endothelial cells of capillaries, mononuclear cells, and fibroblast-like
subepidermal cells in close relationship to the basal membrane is noted
with venous eczema, lipodermatosclerosis, and leg ulcer specimens. Since
TGF-ß inhibits epithelial cell proliferation and migration [27],
its overexpression in venous eczema and lipodermatosclerosis might induce
connective tissue sclerosis and precede leg ulcer development [28].
In summary, the results reported here suggest that alterations in the
expression of EGFR, bFGF and TGF-ß3 precede changes in the affected
skin within progressing stages of CVI. However, the dilemma as to whether
the upregulation of growth factors and their receptors in the pathogenesis
of CVI may cause venous ulceration or whether it is the natural result
of other stimulatory causative factors in circulatory-compromised tissue
still remains an open question, which has to be examined in further studies.
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