Loss of Vascular Endothelial Growth Factor in Human Alopecia Hair Follicles

18S

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

du CROS

hair, whereas subsequent generations of hair have difficulty forming

different stages of the hair cycle of the hairless mutant mice and

because the follicles are distorted and the distal end can become

their normal littermates. Although FGF-2 is known to be a potent mitogen, its localization

separated from the rest of the follicle [5]. Skin samples were obtained from the dorsal region of hairless

to the basement membrane of skin and hair follicles remains

mutant mice and unaffected littermates; the samples were fixed and

unclear, particularly because uncontrolled growth is not observed

paraffin embedded. Tissue sections (5 /Lm) were deparaffinized, then

physiologically in the adult. We therefore hypothesize that FGF-2

M.

is sequestered and held in a latent form by specific heparan sulfate

Reidy, University of Washington, Seattle, WA). A biotinylated goat

proteoglycan molecules in basement membrane of the skin and

anti-rabbit IgG antibody (Cappel) was applied, then the sections

follicles. It is then released in a controlled fashion to receptive

probed with a polyclonal antibody against FGF-2 (a kind gifr of Dr.

incubated with a streptavidin-peroxidase complex (Zymed Laborato­

epidermal cells to initiate proliferation and the invasive processes

ries, Inc.). The sections were developed in

that occur during skin development and during the hair cycle. This

3,3' -diarninobenzidine

hypothesis could explain why differences in FGF-2 expression were

before counterstaining with hematoxylin and mounting. Until 10-14 d of age, mutant animals cannot be distinguished

not observed between hairless mutants and their normal littermates,

from their normal littermates. In newborn skin, FGF-2 expression

because the technique used in this study is not able to distinguish

was found in the basement membrane at the dermal-epidermal

latent basement membrane- bound FGF-2 from released, active

junction and in basement membrane surrounding the

dermal

FGF-2.

Further study of the interactions between FGF-2 and

papillae and the follicles themselves; staining was also found in the

heparan sulfate proteoglycan molecules in the skin is clearly

dennis just beneath the dermal-epidermal junction. Control sec­

required before the specific role of FGF-2 can be elucidated.

tions showed no immunoreactivity. At 17 d of age, the hair follicles of mutant and normal mice were in catagen, mutant follicles becoming very distorted. FGF-2 immunoreactivity remained asso­ ciated with follicle basement membrane but was absent from the

The

finam:ial support of tlte National Alopecia Areata Foundation is gratifully

acknowledged.

basement membrane surrounding the dermal papillae, which were

REFERENCES

no longer incorporated at the bases of the follicles. The expression pattern of FGF-2 did not change during the telogen phase of the hair cycle in mutant or normal mice (20 d of age). At this stage, the hair follicles of the mutant mice were very distorted and many dermal papillae were observed to be stranded in the dennis. By 28

1.

du Cros DL, Isaacs K, Moore GPM: Distribution of acidic and basic fibroblast growth factors in ovine skin during follicle morphogenesis.] Cell Sci 105:667674, 1993

2.

Peters KG, Werner S, Chen G, Williams LT: Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb

d of age, the follicles of the hairless mice remained in telogen whereas those of their notmal littermates had entered anagen of the next cycle. FGF-2 expression did not change in the skin of mutant mice but FGF-2 immunoreactivity was again observed in dermal papillae basement membrane of normal mouse skin. No distinct differences were noted between FGF-2 expression in hair follicles at

fonnation and organogenesis in the mouse. Development 114:233-243, 1992 3.

du Cros DL: Fibroblast growth factor influences the development and cycling of

4.

Crew FAE. Mirskaia L: The character "hairless" in the mouse.} Genet 25:17-24,

5.

Orwin DGF, Chase HB, Silver AF: Catagen in the hairless mouse.

murine hair follicles. Dev Bioi 156:444-453, 1993 1931

Am] Allat

121:489-508, 1967

Loss of Vascular Endothelial Growth Factor in Human Alopecia Hair Follicles Corey K. Goldman, Jui-Chang Tsai, Liliana Soroceanu, and G. Yancey Gillespie Division of Neurosurgery,

V

Department

of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.

ascular endothelial growth factor (VEGF) is the normal product of a single gene that appears to be

or 206 amino acids long. The various polypeptide lengths result from alternative splicing during transcription of the VEGF gene

activated principally by stimuli that are mitogenic

which contains eight exons; the 165 amino-acid isoform is most

for many cell types. VEGF exists in one of four

commonly observed. The VEGF,21 and VEGF'65 isoforms are

different isoforms as a homodimer of disulfide­

secreted in soluble forms from producing cells; the VEGF'89 and

linked polypeptide chains, each of which may be 121, 165, 189,

VEGF206 forms are less soluble because they contain increasing proportions of the hydrophobic carboxy-terminus of the mole­

Reprint University

requests to: Dr. G. Yancey Gillespie, Division of Neurosurgery, of Alabama at Birmingham, THT 65, Birmingham, AL 35294-0006.

cule and after secretion tend to associate with lipid membrane components. VEGF is an endothelial cell (EC) specific molecule binding to

Abbreviations: rtk, receptor tyrosine kinase; VEGF, vascular endothelial

growth factor.

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or KDR receptor tyrosine kinases expressed exclu-

1995 by The Society for Investigative Dermatology, Inc.

VOL.

104, NO.

5, SUPPLEMENT, MAY

Table I.

1995

ALOPECIA HAIR

FOLLICLES DO NOT EXPRESS VEGF

ID1D1unohistocheD1ical Localization ofVEGF in Non-Alopecia Skin" Epidennis

Diagnosis

19S

Case Number

Upper

Mid

Basal

Hair Follicle

Pilosebaceous

Sweat

Capillary

Sw ea t

Meis sner ' s

Gland

Gland

EndotheliuIll

Duct

Corpuscles NV

Normal skin

90-10276

3+

3+

1+

3+

NV

NV

0-2+

NV

Normal skin

85-13326

4+

1+

3+

2-3+

2-3+

1+

NV

NV

Normal skin

91-13 3 12

2+

3+

3+

0

3+

NV

NV

1+

NV

NV

Compound nevus

91-81881

4+

2-3+

1+

2+

1+

4+

4+

4+

3

Seborrheic keratosis

91-13843

4+

2-3 +

0

2+

1+

2+

4+

NV

NV

SqUaInOUS cell ca of scalp

91-11 3 99

4+

3+

0

2-3+

2+

NV

4+

NV

NV

Inflammation

92-91610

3+

2+

0

2-3+

1+

1-2+

0

NV

1-2 +

1-2+

NV

2+

3+

0

2+

2+

4

0

3

91-9938

4+

2+

2+

NV

Contact dermatitis

84-1190

Normal skin

89

Normal skin

-

1 2 88 5

NV

1+

0

NV

0

0

0

3+

1+

NV

NV

0

Normal skin

84-92 2 8

2-3+

2-3+

1+

2+

2+

1+

2-3+

1+

2-3+

Normal skin

90-7325

1-2+

2+

1+

1+

0

NV

4+

NV

Connective tissue nevus

91-2 2 88

1+

0-1+

0-1+

1+

NV

0

NV

NV

4+

•

NV

Grading criteria: 0, negative; 1 +, equivocal; 2+. light staining; 3+, moderate staining; 4+, intense staining. NY, none visualized.

sively on endothelial cells. VEGF induces EC proliferation (angiogenesis), increases vascular permeability, and enhances EC-mediated hyper-coagulability by promoting thromboplastin activation and increasing the expression of von Willebrand Factor (factor VIII-related antigen) on the surfaces of EC. It is believed that the different isoforms of VEGF mediate each of these EC responses to a different extent, e.g., VEGF,2, has principally angiogenic properties whereas VEGF,65 has angio­ genic and vascular permeability activities. Here we present immunohistochemical evidence that VEGF has virtually disappeared from hair follicles in alopecia areata and to a lesser extent in androgenic alopecia. Loss of the ability of hair follicle cells to produce this potent EC-specific growth factor probably has profound effects on maintenance of proper vascular support of skin and its organelles, which could diminish normal follicle cell function. Rabbit

antibody

to

recombinant

human

hematoxylin and eosin f or standard histologic diagnosis.

All

diagnoses were

confirmed by dermatologists at the University of Alabama at Birmingham. Punch bi opsies obtained as part of routine diagnosis for alopecia areata by Dermatology Clinic physicians at the University were accessed as coded paraffin-embedded specim.ens through the University Tissue Procurement Facility (Department of Pathology). Punch-biopsy specimens from 20 normal male volunteers with androgenic alopecia were obtained by physicians of d,e Latham Hair Clinic (Birmingham,

AL). Each

patient donated bald and haired

skin saInples. Selected archival surgical specin:tens were reviewed to identifY those containing normal skin structures

and froIll

these several representative

saInples were chosen at randoIll as non-alopecia tissues.

Inununohistochemical Analyses

Post-fixed sections were exposed to

either rabbit or mouse monoclonal antibodies (60 min, room temperature) and washed prior to incubating

with

th e appropriate anti-globulin reagent.

Antigen-dependent color development was achieved by incubating reacted tissue samples either with diaminobenzidine (10 f.Lg/rul in 0.05 M Tris, pH 7.2 ) for horseradish peroxidase- coupled reagents or with

MATERIALS AND METHODS

Antibodies

each specin:ten were prepared and representative slides were stained with

mixture

of

nitroblue tetrazolium (330 f.Lg/ml in 0.1 M Tris, pH 9.5) and bis-chloro­ VEGF'65

(rh­

indozolium phenol (165 f.Lg/ml) for alkaline phosphatase- conjugated re­

VEGF,6,) was raised by nine successive immunizations with 50-200 f.Lg

agents. Tissues were counterstained with Mayer's hematoxylin (DAB) or

antigen. Rabbit antisera were adsorbed with immobilized fetal bovine

nuclear fast red (NBT/BCIP), dehydrated, and Illounted in Eukritt medium

serum and iIllIllobilized human serum and shown to be specifically blocked

in

binding to VEGF

in

human tumor and placental tissue saInples by

pre-incubation with rhVEGF'65' Monoclonal antibody A4.6.1 was obtained

for microscopic inspection and photomicrography. Grading of the intensity of iIllmunostaining for specifically identified tissue organelles or structures was judged independently by at least two different trained professionals and

in purified form as a kind gift ofDr.JinKiIn (Genentech, Inc.). Monoclonal

was based on a scale of 0 to 4, where 0 is " none "

antibody SC3.F8 was produced in our laboratory by immunizing BALB/c

"light", 3 is "Illoderate," and 4 is "intense." Values were obtained for at

mice with Illultiple sequential injections of purified human glioma-derived

least four different high-pow e r (45X) fields of view.

,

1

is "equivocal", 2 is

VEGF and a VEGF peptide based on the putative binding site sequence. Hybridomas

from several fusions were screened and one,

designa ted

5C3.FS (an IgG1), was chosen based on antigen recognition and avidity. Biotinylated horse anti-mouse immunoglobulin antibody or bi otinylated goat anti-rabbit immunoglobulin (Vector Laboratories, Burlingame, CA) were purchased and used according to manufacturers' directions with

avidin-biotin horseradish peroxidase or avidin-biotin alkaline phosphatase to localize primary antibody binding to tissues. Rabbit sera was used to prepare a purified rabbit IgG fraction by protein G affinity chroIllotography. Rabbit immunoglobulin concentratiou was adjusted to 2 00 f.Lg/rul in phosphate-buffered saline. Mouse IgG2a or IgGl immunoglobulins were purchased from Southern Biotechnology, Inc.

(BirminghaIn,

AL)

at a

concentration of 1 mg/ml. Monoclonal antibody 2 9.1.1 (Sigma Chemical Co.) was used to stain for epidermal growth factor expression in these tissues.

Tissue Specimens

All

tissue samples were fixed in 10% neutral buffered

formalin or 4 % paraformaldehyde and embedded in paraffin for sectioning. Sections 8-10-f.Lm thick were mounted on glas s slides coated with

1%

3 -aminopropyl-triethoxysilane in acetone, de-paraffinized by successive exposures to xylene and alcohol rinses, rehydrated to ph osphate-buffer ed

saline, and post-fixed by im.mersion for 30 min in 0.01 M sodium citrate, pH

4.0, with initial

heating to 95°C by microwave. Multiple serial sections frOIll

RESULTS AND DISCUSSION In non-alopecia individuals, upper (average score 3.11 +) and middle (average 2.46+) epidermal layers showed 2-4+ staining whereas the basal levels had 0-2+ (average 0.26+) staining intensity (Table I). In contrast, patients with AA showed greatly diminished staining intensity (0-3+) in upper (average 2.6+) and middle (average 1.7 +) layers. There was no detectable staining in basal epidermis (Table II). Patients with androgenic alopecia showed intermediate levels. Most prominent was the intense staining (average 2.62+) seen in the cells lining the hair follicles in non-alopecia individuals, which was noticeably re­ duced to absent in patients with alopecia (average 1.3+). Likewise, VEGF was significantly reduced in pilosebaceous and sweat glands in the skin of AA patients compared with normal individuals' skin organelles. These data suggest that the reduc­ tion of VEGF production by hair follicles may lead to a loss of vascular support to affected skin. With subsequent inflammatory changes resulting from inadequate tissue vascularization, this could lead to atrophy of important skin organelles, especially

20S

GOLDMAN

THE

ET AL

Table II.

Itnmunohistochemical Localization of VEGF in Alopecia Skin" Epidermis

Diagnosis

JOURNAL OF INVESTIGATIVE DERMATOLOGY

Hair

Sweat

Capillary

Sweat

Meissner's

Basal

Follicle

Gland

Gland

Endothelium

Duct

Corpuscles

0

0

0-1+

1+

1-2+

4+

0

1+

4+

4+

0

2-3+

2+

3+

0

3+

NV

85-3560

1+

0

0

1+

0-1+

2+

4+

NV

NV

91-7303

2+

1+

0

1+

1+

1+

0

0

0

92-18181

0

0

0

NV

1+

NV

0

0

86-10962

1+

0

0

0

1+

NV

4+

91-18977

Oil +

0

0

0-1+

0

0

4+

0

90-11704

4+

3- 4+

0

1-2+

N.V.

1+

2+

NV

3- 4+

0-1+

0-1+

0

0-1+

0-1+

1+

1+

1-2+

NV

Case Number

Upper

92-173 62

3+

87-00847

Mid

Pilosebaceous

Alopecia areata Hot comb alopecia Alopecia areata Alopecia areata (regrowth) Alopecia (scarring) Alopecia areata Androgenic alopecia

NV

Androgenic alopecia Alopecia areata a

83-6451

Grading criteria: 0, negative; 1 +, equivocal; 2+, light staining; 3+, moderate staining; 4+, intense staining. NV, none visualized.

hair follicles. This hypothesis is supported by the finding that loss

growth

of capillaries are early changes in alopecia and revascularization

production of

factor,

and

VEGF

fibroblast

growth

factor)

can

stimulate

in cells of neuro-ectodermal origin, it

precedes hair regrowth. Given that a number of distinct growth

would be important to assess whether or not cells lining hair

factors

follicles retain responsiveness to these growth factors.

(notably

epidermal

growth

factor,

platelet-derived

Analysis of Gene Expression in Isolated Single Hair Follicles: An Approach Using Semiquantitative Reverse-Transcriptase-Polymerase Chain Reaction Rolf Hoffmann and Rudolf Happle University,

Department of Dermatology, Philipp

air

H

in vitro

Marburg, Germany

biology is an important topic of cutaneous

research. After several decades of hair research,

of gene expression in single hair follicles. As interferon

(IFN)-)' has

been shown to induce intercellular adhesion molecule (ICAM)-l

however, most of the basic mechanisms have

expression on hair follicle epithelial cells [1], we used this phenom­

remained unexplained. Our lack of understanding

enon in short-term cultured anagen hair follicles to establish our

hair biology is due in part to the lack of appropriate

system.

models. Hair growth is a dynamic, highly coordinated

Intact, viable anagen hair follicles were isolated by microdis­

process that involves the expression of a variety of different genes,

section as described previously [2] and kept in culture with or

but so far no appropriate in "jim system has been described that

without recombinant human

allows precise determination of specific mRNA expression within a

RNA from individual hair follicles was isolated [3] and reverse

single hair follicle. Consequently, the purpose of the present study

transcribed by the use of hexamer primers and murine moloney

was to establish an ill

"itro

assay for semiquantitative measurement

IFN-)'.

After incubation,

total

tumor virus reverse-transcriptase. The polymerase chain reac­ tion (PCR) was performed with specific primers for f)-actin and

Reprint requests to: Dr. Rolf Hoffinann, Department of Dermatology, Philipp University DeutschhausstraBe 9, 35033 Marburg, Germany.

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ICAM-1. The PCR setup was optimized to obtain only one PCR product of the expected length. Specificity of each amplified

,

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1 995 by The Society for Investigative Dermatology, Inc.