Characterization of Prostaglandin F 2α Receptors in Human Eyelids

Abstract

Purpose

Elongation, thickening, and crowding of eyelashes are commonly seen after topical use of prostaglandin analog eyedrops for glaucoma treatment. The purpose of this study was to demonstrate the presence and characterize the location of prostaglandin analog F_2α receptors (PGF_2α) in human hair follicles.

Methods

In this observational clinical laboratory study, excised eyelid specimens following eyelid surgery were studied. Resected portions of eyelids were submitted for histopathologic evaluation. For immunohistochemistry evaluation, a polyclonal antibody directed against PGF_2α was purchased from Cayman Chemical. The staining procedure was carried out on an automatic stainer.

Results

Out of 26 patients recruited, final analysis was conducted on 17 eyes of 15 patients. There were 10 men and 5 women (mean age 77 ± 14 years). Staining was detected only in hair follicles in the anagen stage (37 slices). No variation in pattern, distribution, or intensity of immunostaining was noted among sections of different individuals. Only the bulb and stem of the hair follicle stained positive. In the bulb, the strongest staining occurred in the matricular cells and in the inner sheath layer. In the stem, the strongest staining occurred in the Huxley layer of the inner sheath.

Conclusions

This immunohistologic study found that PGF_2α receptors were located predominantly in the inner root sheath of the bulb and stem of eyelashes and expressed only in eyelashes in the anagen phase.

Keywords

Eyelids Hair follicle Human Receptors

Introduction

Prostaglandin analogs have a prominent role in intraocular pressure reduction in ocular hypertension as well as glaucoma (1). Due to their potency and the lack of systemic side effects, they are often considered as first-line therapy (2). However, local side effects have been reported to a certain degree with all prostaglandin analogs. Elongation, hypertrichosis, and hyperpigmentation of the eyelashes are well-described side effects of topical prostaglandins and prostamide analogs that may sometimes reach blemishing levels (3-8). Iris and periocular hyperpigmentation, periorbital muscle atrophy, and both periorbital and orbital lipodystrophy may also occur (9-12). A variety of receptors for prostaglandin analogs in human tissues have been studied (EP_1-4, PGE₂, PGF_2α, and others) (13, 14). Their presence throughout the human scalp hair follicle, specifically in the dermal papilla outer root sheath, has been proven (14, 15). The capability of PGF_2α and FP receptors to introduce telogen to anagen transition in hair follicles has been documented (16).

Studies that have researched the EP and PF receptors in ocular tissues (cornea, trabecular meshwork, iris, ciliary body, choroid, and retina) documented a wider distribution of EP receptors (13). No studies have investigated and characterized the location of these receptors in human eyelid hair follicles. As the prostaglandin analog effect in the eyelid is mainly mediated via PGF_2α (16), the purpose of this study was 1) to demonstrate the presence of PGF_2α in human hair follicles; 2) to characterize their distribution along the hair follicle and its variation in different growth stages; and 3) to investigate the occurrence of heterogeneity in receptor expression among patients.

Methods

All data for the study were collected and analyzed in accordance with the policies and procedures of the institutional review board of Meir Medical Center and the tenets set forth in the Declaration of Helsinki.

A prospective consecutive design was used. The specimens studied were collected from patients undergoing lateral tarsal strip dissected as part of an ectropion repair procedure. When operated, none of the patients had a history of prostaglandin analog treatment or signs of inflammatory lesions or clinically appreciable signs of inflammation of the eyelids themselves. All the patients included in this study were Caucasian. For the sake of consistency, all the specimens were collected from the lower eyelid. Only specimens in which at least one hair follicle was identified were included in this study.

Histologic preparation

The resected portions of eyelids were immediately fixed in 4% buffered formalin and submitted for histopathologic processing and evaluation, according to routine protocols. Tissue sections of 4-μm thickness, stained by hematoxylin & eosin, were prepared from paraffin blocks.

Immunohistochemistry

The FP receptor polyclonal antibodies, directed against the PGF_2α receptor, were purchased from Cayman Chemical (Ann Arbor, Michigan, USA; catalog no. 101802), as described and validated by previous studies (17-21). The staining procedure was carried out on a Ventana Benchmark automatic stainer (manufacturer: Ventana, Tucson, Arizona, USA). The FP receptor antibodies were diluted to 1:1100. The primary antibody was detected by streptavidin, which was conjugated to a biotinilated secondary antibody. The FP receptor and antibody complex was expressed as a red staining in the tissue sections.

Histopathologic review

All specimens were reviewed by the pathologist (D.K.) and staining was graded using a qualitative scale ranging between 0 and 2:0 = light staining, similar to baseline staining observed in the conjunctival epithelium; 1 = moderate staining; and 2 = intense staining.

Results

Overall, 26 patients were recruited for this study. Final analysis was conducted on 37 slices examining 17 eyes of 15 patients in which at least one hair follicle was identified. There were 10 men and 5 women. The mean ± SD age of the patients was 77 ± 14 years. The number of slices studied per patient varied from 1 to 7 with a median of 3. The slices were taken from the right eye in 7 patients, left eye in 6 patients, and both eyes in 2 patients.

All specimens contained hair follicles in the anagen phase while hair follicles in the catagen phase were detected in 4 specimens only. Staining was detected only in hair follicles in the anagen stage. No variation in pattern, distribution, and intensity of immunostaining was noted among sections of different individuals. Table I depicts the immunolocalization and grade of the PGF_2α receptors in the hair follicles.

Table I

Location and degree of PGF_2α receptor staining in hair follicles

Segment of hair follicle	Outer sheath	Inner sheath	Matricular cells
Bulb	0	2	2
Stem	1	Cuticle/Henle/Huxley 0/0/2	0
Isthmus	0	NA	NA
Infundibilum	0	NA	NA

NA = not applicable.

Of the 4 parts of the hair follicle (bulb, stem/suprabulbar, isthmus, and infundibulum), only the first 2 stained positive for the receptors. In the bulb, the strongest staining occurred in the matricular cells and in the inner sheath layer. No staining occurred in the outer sheath of the bulb (Fig. 1).

Fig. 1

A longitudinal section through hair bulb and stem. There is strong cytoplasmic staining of the inner sheath (INS) and of the matricular cells (MC). Note that there is no staining in the outer sheath (OS).

The inner sheath divides into 3 apparent layers at the stem (suprabulbar) level (Henley, Huxley, and cuticle). The presence of PGF_2α receptors was observed mainly in the Huxley layer, while no staining occurred in the Henley layer. Faint staining occurred in the outer sheath cells of the stem (Fig. 2).

Fig. 2

A transverse section of hair follicle at level of stem. Intense (grade 2) red staining in Huxley's layer of inner sheath (wide arrows). No staining in Henle's layer of inner sheath (thin arrow). Faint (grade 1) cytoplasmic staining of outer sheath cells (OS).

In general, whenever staining was detected, it occurred mostly in the cytoplasm of the cells with slight membranous staining. In the matricular cells, due to the paucity of cytoplasm, it was hard to determine whether this staining was solely cytoplasmic or also perinuclear.

Discussion

In this study, we demonstrated the presence of PGF_2α receptors in human eyelid hair follicles. Immunohistochemical staining for PGF_2α receptors was noted in the bulb and stem segments in hair follicles at the anagen phase. Staining occurred mostly in the cytoplasm of the cells. No heterogeneity in receptor expression was demonstrated among patients.

The PGF_2α receptor is a membranous receptor. In our study, staining of the PGF_2α receptor occurred mostly in the cytoplasm. This is in concordance with findings by Schlötzer-Schrehardt et al (13), who reported that staining of the receptor in human ocular tissues occurred in the cytoplasm. They reported that the most prominent localization of the staining occurred in the nuclear envelope and occasionally on outer mitochondrial membranes, on rough endoplasmic reticulum, and in cytoplasmic vesicles.

While there is abundant evidence on the effect of prostaglandin analogues on the growth of eyelashes, there is a paucity of histologic studies profiling the location of the receptors. To date, one cannot predict which patient will develop the adverse side effects. Even brief exposure to an ophthalmic prostaglandin analog can be associated with eyelash change (3). In fact, bimatoprost solution 0.03% (Latisse; Allergan, Inc., Irvine, California, USA) was found to be effective in enhancing eyelash growth (22).

This is the first study we are aware of to investigate the distribution of PGF_2α receptors in the hair follicles of human eyelids. Schlötzer-Schrehardt et al (13) demonstrated the expression and localization of FP receptor proteins by immunohistochemistry in 10 normal human donor eyes. No comment was made on the expression of the receptor in the hair follicles. Colombe et al (15) demonstrated, on 3 scalp hair biopsies, that FP expression was essentially restricted to the outer root sheath companion layer and dermal papilla, and speculated that this could explain the biological effect of PGF_2α on hair regrowth. Whether the difference in locations of staining (outer sheath versus inner sheath) between the 2 studies may be attributed to the fact that these are 2 different types of hair (scalp hair versus eyelashes) is unclear.

Our findings of staining of PGF_2α receptors, preliminary in hair follicles in anagen stage, are in agreement with the understanding that hair growth occurs at this stage (23).

In this study, no apparent diversity in the degree of hair follicle staining among the specimens was noted. There are several possible explanations for this observation. This may be a result of the relatively small number of specimens. Another possible explanation is the lack of diversity in the patients; none was being treated with prostaglandin analogs and none had hypertrichosis (24, 25). The lack of variation may also be a result of the older age of our study group. Although the range was 42 to 94 years, the mean age was 77.

The hypertrichosis of eyelashes associated with prostaglandin and prostamide analogs usually occurs together with darkening of the eyelashes. This staining technique used did not efficiently evaluate the staining of the melanocytes as previous bleaching was not performed. Future studies should characterize the PGF_2α staining of melanocytes.

The small number of specimens as well as the method of staining used precludes us from quantitative evaluation of PGF_2α receptors.

In this study, all specimens were taken from lower eyelid specimens dissected as part of an ectropion repair procedure. Previous studies have demonstrated that the upper eyelashes are both longer and more numerous than their lower counterparts (26, 27). One study demonstrated nearly 2 times the amount of lashes in the upper eyelid versus the lower eyelid (28). Therefore, conclusions from this study may not fully apply to lashes from the upper eyelid. In addition, except for the mildest cases, ectropion is connected with exposure of conjunctiva, which causes inflammation. This was dealt with by only including specimens from patients with no clinically apparent signs of inflammation; however, subclinical inflammation may have affected the expression of prostaglandin receptors in this study (29, 30).

In this study, none of the patients was being treated with prostaglandin analogs. Future studies should compare the staining in eyes treated with prostaglandin analogs to those not being treated with prostaglandin analogs.

This study focused on the location of the receptors and did not offer insight into the function of PGF_2α in human eyelid hair follicle growth. Further studies should focus on this issue.

In summary, we found that prostaglandin PGF_2α receptors were located predominantly in the inner root sheath of the eyelashes and expressed in eyelashes at the anagen phase.

Footnotes

Financial support: No financial support was received for this submission.

Conflict of interest statement: None of the authors has conflict of interest with this submission.

Meeting presentation: Presented at the 23^rd annual meeting of the American Glaucoma Society, San Francisco, California, USA, March 1, 2013.

References

Digiuni

Fogagnolo

Rossetti

A review of the use of latanoprost for glaucoma since its launch. Expert Opin Pharmacother 2012; 13: 723–45

Denis

Baudouin

Bron

First-line latanoprost therapy in ocular hypertension or open-angle glaucoma patients: a 3-month efficacy analysis stratified by initial intraocular pressure. BMC Ophthalmol 2010; 104

Johnstone

Hypertrichosis and increased pigmentation of eyelashes and adjacent hair in the region of the ipsilateral eyelids of patients treated with unilateral topical latanoprost. Am J Ophthalmol 1997; 124: 544–7

Tosti

Pazzaglia

Voudouris

Tosti

Hypertrichosis of the eyelashes caused by bimatoprost. J Am Acad Dermatol 2004; 51 SupplS149–50

Hempstead

Unilateral trichomegaly induced by bimatoprost ophthalmic solution. J Drugs Dermatol 2004; 3: 571–2

Herane

Urbina

Acquired trichomegaly of the eyelashes and hypertrichosis induced by bimatoprost. J Eur Acad Dermatol Venereol 2004; 18: 644–5

Hart

Shafranov

Hypertrichosis of vellus hairs of the malar region after unilateral treatment with bimatoprost. Am J Ophthalmol 2004; 137: 756–7

Elgin

Batman

Berker

Ilhan

The comparison of eyelash lengthening effect of latanoprost therapy in adults and children. Eur J Ophthalmol 2006; 16: 247–50

Kapur

Osmanovic

Toyran

Edward

Bimatoprost-induced periocular skin hyperpigmentation: histopathological study. Arch Ophthalmol 2005; 123: 1541–6

10.

Tsai

Sivak-Callcott

Haik

Zhang

McLean

Latanoprost-induced iris heterochromia and open-angle glaucoma: a clinicopathologic report. J Glaucoma 2001; 10: 411–3

11.

Sira

Verity

Malhotra

Schlötzer-Schrehardt

Zenkel

Nüsing

Topical bimatoprost 0.03% and iatrogenic eyelid and orbital lipodystrophy. Aesthet Surg J 2012; 32: 822–4

12.

Wang

Koh

Cheng

Periorbital muscle atrophy associated with topical bimatoprost therapy. Clin Ophthalmol 2014; 8: 311–4

13.

Schlötzer-Schrehardt

Zenkel

Nüsing

Expression and localization of FP and EP prostanoid receptor subtypes in human ocular tissues. Invest Ophthalmol Vis Sci 2002; 43: 1475–87

14.

Colombe

Vindrios

Michelet

Bernard

Prostaglandin metabolism in human hair follicle. Exp Dermatol 2007; 16: 762–9

15.

Colombe

Michelet

Bernard

Prostanoid receptors in anagen human hair follicles. Exp Dermatol 2008; 17: 63–72

16.

Sasaki

Hozumi

Kondo

Influence of prostaglandin F2alpha and its analogues on hair regrowth and follicular melanogenesis in a murine model. Exp Dermatol 2005; 14: 323–8

17.

Qualtrough

Kaidi

Chell

Jabbour

Williams

Paraskeva

Prostaglandin F(2alpha) stimulates motility and invasion in colorectal tumor cells. Int J Cancer 2007; 121: 734–40

18.

Suzuki-Yamamoto

Toida

Sugimoto

Ishimura

Colocalization of prostaglandin F(2alpha) receptor FP and prostaglandin F synthase-I in the spinal cord. J Lipid Res 2009; 50: 1996–2003

19.

Guo

Kasaraneni

Sun

Myatt

Cross talk between PKC and CREB in the induction of COX-2 by PGF2α in human amnion fibroblasts. Endocrinology 2012; 153: 4938–45

20.

Wånggren

Stavreus-Evers

Olsson

Andersson

Gemzell-Danielsson

Regulation of muscular contractions in the human Fallopian tube through prostaglandins and progestagens. Hum Reprod 2008; 23: 2359–68

21.

Blesson

Büttner

Masironi

Sahlin

Prostaglandin receptors EP and FP are regulated by estradiol and progesterone in the uterus of ovariectomized rats. Reprod Biol Endocrinol 2012; 10: 3

22.

Woodward

Haggerty

Stinnett

Williams

Bimatoprost 0.03% gel for cosmetic eyelash growth and enhancement. J Cosmet Dermatol 2010; 9: 96–102

23.

Stenn

Paus

Controls of hair follicle cycling. Physiol Rev 2001; 81: 449–94

24.

Woodward

Regan

Lake

Ocklind

The molecular biology and ocular distribution of prostanoid receptors. Surv Ophthalmol 1997; 41 Suppl 2S15–21

25.

Abramovitz

Boie

Nguyen

Cloning and expression of a cDNA for the human prostanoid FP receptor. J Biol Chem 1994; 269: 2632–6

26.

Moses

The eyelids. In: Adler's Physiology of the Eye: Clinical Application, 5th ed.

St. Louis

CV Mosby

1970: 1–16

27.

Elder

Anatomy and physiology of eyelash follicles: relevance to lash ablation procedures. Ophthal Plast Reconstr Surg 1997; 13: 21–5

28.

Freitag

Lee

Johnstone

Sires

Retrospective review of eyelash number in patients who have undergone full-thickness eyelid resection. Ophthal Plast Reconstr Surg 2014; 30: 1–6

29.

Stefanyszyn

Hidayat

Flanagan

The histopathology of involutional ectropion. Ophthalmology 1985; 92: 120–7

30.

Ueta

Sotozono

Yokoi

Inatomi

Kinoshita

Prostaglandin E receptor subtype EP3 expression in human conjunctival epithelium and its changes in various ocular surface disorders. PLoS One 2011; 6 e25209.