Arch Dermatoi Res (1992) 284:246 249
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rlNl~ rl
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,9 Springer-Verlag 1992
Identification of a cell layer containing a-smooth muscle actili in the connective tissue sheath of human anagen hair A. Urabe, M. Furumura, S. Imayama, J. Nakayama, and Y. Hori Department of Dermatology, Kyushu University, Faculty of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan Received November 12, 1991
Summary. Immunohistochemical and immunoelectron microscopy studies revealed the presence of m-smooth muscle (~-SM) actin in fibroblasts located in the connective tissue sheath (CTS) of human anagen hair follicles. Immunostaining was positive from the base of the bulb to the upper part of the lower portion of the mature anagen hair follicles. The late catagen hair follicles did not stain. Ultrastructurally, ~-SM actin was detected only in the fibroblasts located in the innermost layer of the transverse collagenous fibres. Since ~-SM actin is located in cells with contractile potential, this newly identified layer may play an important role in the morphological changes of the lower portion of the hair follicle during the hair growth cycle. Key words: H a i r - C o n n e c t i v e tissue s h e a t h - A l p h a S m o o t h m u s c l e actin - I m m u n o h i s t o c h e m i s t r y munoelectron microscopy
Im-
T h e c o n n e c t i v e tissue s h e a t h (CTS) is a c o m p o n e n t of the h a i r follicle. U l t r a s t r u c t u r a l l y , the C T S c o m p r i s e s t h r e e layers: (1) the i n n e r c o l l a g e n layer, c o m p o s e d of c o l l a g e n fibres r u n n i n g p a r a l l e l to the h a i r l o n g axis: (2) the m i d d l e c o l l a g e n layer, c o m p o s e d of t r a n s v e r s e collagen fibres a n d i n t e r m i n g l e d f i b r o b l a s t s ; a n d (3) the o u t e r c o l l a g e n layer, c o m p o s e d of c o l l a g e n fibres r u n n i n g in v a r i o u s d i r e c t i o n s a n d cellular c o n s t i t u e n t s such as fibroblasts, b l o o d vessels a n d fat cells [2, 3]. I m m u n o h i s t o chemically, several a n t i b o d i e s h a v e b e e n used to identify e p i t h e l i a l c o m p o n e n t s of h a i r follicles, b u t specific m a r kers for the C T S h a v e n o t b e e n found. A l p h a - s m o o t h m u s c l e ( ~ - S M ) actin is a d i f f e r e n t i a t i o n m a r k e r o f s m o o t h m u s c l e cells a n d is p r e s e n t in all s m o o t h m u s c l e cells a n d p e r i c y t e s [8]. W e m a d e use o f the a n t i - ~ - S M actin a n t i b o d y to o b s e r v e the presence o f this a c t i n in f i b r o b l a s t s in the C T S o f h u m a n a n a g e n hairs. P o s s i b l e f u n c t i o n s o f these cells in a s s o c i a t i o n w i t h the h a i r g r o w t h cycle a r e discussed.
Correspondence to: A. Urabe
Materials and methods
Immunohistochemistry Specimens of normal adult scalp skin were obtained from five Japanese patients (three males and two females, 53-72 years old) during surgical removal of benign turnouts. The specimens were snap-frozen in liquid nitrogen and stored at - 8 0 ~ until use. Serial frozen sections (5 gin) were placed on microscopic slides coated with poly-L-lysine (Sigma Chemicals, St. Louis, USA), airdried and fixed in cold acetone for 10min. To confirm the distribution of ~-SM actin in various stages of the hair growth cycle, two patients (males aged 13 and 51 years) with congenital melanocytic naevus were selected, from whom a H & E-stained sections showed late catagen and early anagen hair follicles. Paraffin blocks of these were recur, and 5 gm paraffin sections were de-waxed in xylene and washed in ethanol. After blocking with 2% normal horse serum, both frozen and paraffin sections were incubated overnight at 4 ~ with anti-~-SM actin antibody (Sigma Chemicals) at a dilution of 1:2000 using 0.1% bovine serum albumin (BSA) in PBS as diluent. Following incubation with biotinylated anti-mouse IgG (Immunotech, Marseille, France) at a dilulion of 1 : 40, the sections were reacted with the fluorescein-conjugated streptavidin-biotin complex (Gibco BRL, Gaithersburg, USA) at a dilulion of 1:40. Both incubations were performed for 45 min at 37 ~ The sections were examined under a Zeiss (Oberkochen, FRG) fluorescent microscope. H & E staining of serial frozen sections was carried out using conventional procedures.
Electron microscopy Specimens were fixed in 3% glutaraldehyde and post-fixed in 1% osmium tetroxide in 0.1M cacodylate buffer. The tissues were dehydrated in increasing concentrations of ethanol and embedded in Epon 812. Ultrathin tissue sections were stained with uranyl acetate and lead citrate.
Immunoelectron microscopy Specimens were fixed in periodate-lysine-paraformaldehyde,dehydrated in increasing concentrations of dimethyl formamide in water, and embedded in Lowicryl K4M at 4 ~ (Low; Waldkraiburg, FRG) as previously described [1]. Ultrathin sections, collected on nickel grids, were incubated in a drop of 1% BSA in PBS for 30 min and then incubated in anti-c~-SM actin antibody at a dilution of 1 : 100 for 1 h. After rinsing with PBS, the sections were incubated
247 in goat anti-mouseIgG labelled with 15 nm gold particles (GAMG15; Janssen, Olen, Belgium)at a dilution of 1:20 for 1 h, washed again thoroughly with water, and stained with uranyl acetate and lead citrate. For the control, the complete staining protocol was followed, but without incubation in the primary antibody. All specimens were examined under a Hitachi (Tokyo,Japan) H-7000 electron microscope.
Results
Histologically, the lower portion of the anagen hair follicle was composed of the hair, the inner root sheath, the outer root sheath, the CTS including the vitreous membrane and the dermal papilla (Figs. 1A, 2A). In late catagen, a thin epithelial cord and dermal papilla were present at the base of the follicle (Fig. 4A). Dilated capillaries were seen below the papilla. In early anagen, a newly formed hair follicle descending from the epithelial sac with club hair was observed (Fig. 5A). A high-power view of the section showed two or three layers of fibroblasts with small nuclei surrounding the vitreous membrane (Fig. 5B).
The localization of ~-SM actin was observed in vascular smooth muscle cells, pericytes and myoepithelial cells of eccrine glands in the normal human scalp skin. In mature anagen hair follicles present in all sections examined, a clear staining was seen in the CTS (Fig. 1 B) as well as arrector pili muscle. The positive reaction was observed in the bulb of the anagen hair follicles, but there was no staining in the dermal papilla (Fig. 2B). The staining faded at the upper part of the lower portion of the hair follicles and was no longer present at the arrector pili muscle (Fig. 3). No reaction was seen in the connective tissue around the hair follicles at the level of the isthmus and the infundibulum. There was no staining in the late catagen hair follicle, except for vascular smooth muscle cells and pericytes (Fig. 4B). In early anagen, a weak positive staining was observed in the CTS (Fig. 5 C). Ultrastructurally, the CTS of a mature anagen hair follicle was composed of a basal lamina and surrounding collagen tissue which was further separated into three distinct layers, the inner, intermediate and outer collagen layers, as noted by other investigators [3]. Fibroblasts located in the innermost layer of the intermediate layer are characterized bY their ramified configuration, relatively dense cytoplasm filled with fine filaments and a few cytoplasmic organellas (Fig. 6). Immunoelectron microscopy revealed that anti-~-SM actin antibody labelled the cytoplasm of these fibroblasts (Fig. 7) but not the fibroblasts located in the outer area of the intermediate layer and in the outer collagen layer. Pericytes surrounding the small vessels in the CTS were also labelled with this antibody. Discussion
Fig. 1A. The lower portion of mature anagen hair follicle (H & E, 90). B The CTS stained with anti-c~-SMactin antibody (100) Fig. 2A. The bulb of mature anagen hair follicle (H & E, 90). B Note the lack of staining of the dermal papilla (100)
We obtained evidence that ~-SM actin is present in fibroblasts located in the innermost layer of the transversely running collagen fibres of the CTS. Since ~-SM actin has previously been found only in cells with contractile function [7] and the presence of this actin was confined to the ower portion of the hair follicles, it seems reasonable to speculate that these cells play an important role in the hair growth cycle involving dynamic kinetics of the lower portion of the hair follicles. The hair cycle is composed of three different stages; i.e. anagen (proliferative phase), catagen (subsiding growth) and telogen (resting phase). At the beginning of catagen, the club-shaped, keratinized proximal shaft is pushed upwards by a column of epithelial cells, and subsequently the dermal papilla moves upwards as the epithelial column shortens [4]. Montagna and Parakkal [6] found that the collagen fibres in the CTS were broken down during the process by the tightly packed layer of macrophages, and they speculated that macrophages participated in the degenerative changes of the CTS. However, Ito and Sato [3] recently demonstrated that macrophages did not play a specific role in the dynamic changes observed in catagen, and they speculated that the CTS around the lower end of the epithelial cord was digested by enzymes produced by the epithelial cells. We
248
Fig. 3. The upper part of the lower portion of the mature anagen hair. A positive staining of the CTS (arrowhead) discontinuouswith that of the arrector pill muscle (arrow) (50) Fig. 4A. The late catagen hair follicle in paraffin section (H & E, 90). B Note the lack of staining around the hair follicle, except for vascular smooth muscle cells and pericytes (100) Fig. 5A. The early anagen hair follicle in paraffin section. The telogen club hair (T) was present within its epithelia1 coating (H & E, 45). B High-power view of the early anagen hair follicle (H & E, 180). C The CTS of the early anagen hair follicle was weakly stained with the antibody (200)
Fig. 6. Electron micrograph of the CTS of the mature anagen hair. The inner collagen layer (a), located next to the basal lamina (arrowheads), was composed of collagen fibres running parallel to the long axis of the hair. The intermediate layer (b) was composed of collagen fibres running transversely against the hair axis and fibroblasts (*). The outer layer (c) consisted of irregularly arranged
collagen fibres intermingled with fibroblasts and small blood vessels (bar, 1 ~tm) Fig. 7. Electron micrograph of the fibrobIasts immunolabelled with anti-c~-SM actin antibody. Gold particles are distributed in the cytoplasm (bar, 0.1 p,m)
249 propose that retraction of the epithelial cord and the basal lamina m a y be caused by the contractile function of the fibroblasts present in the innermost layer of the CTS. Ultrastructurally, both the basal lamina and the inner collagen layer of the CTS present outside the basal lamina showed a wavy appearance during catagen [3]. This morphological change supports the notion of a contractile function of these cells present around the inner collagen layer. The expression of ~-SM actin by fibroblasts in the CTS m a y be useful for evaluating histogenesis of tumours considered to be of hair follicle origin. This anti-~-SM actin antibody m a y also be of use for analysing the origin of cultured hair follicle cells, since plucked anagen hairs sometimes contain the CTS [5]. The newly identified thin layer composed of fibroblasts stained with anti-~-SM actin antibody described here probably has a characteristic function and studies to identify this putative function are underway.
Acknowledgement. The authors thank M. Ohara for critical comments on the manuscript.
References 1. Altman LG, Schneider BG, Papermaster DS (1984) Rapid embedding of tisses in Lowicryl K4M for immunoelectron microscopy. J Histochem Cytochem 32:1217-1223 2. Hashimoto K (1981) The environment of the hair follicles. In: Orfanos CE, Montagna W, Stuttgen G (eds) Hair research. Springer, Berlin, Heidelberg, New York, pp 172-182 3. Ito M, Sato Y (1990) Dynamic ultrastructural changes of the connective tissue sheath of human hair follicles during hair cycle. Arch Dermatol Res 282:434-441 4. Kligman AM (1959) The human hair cycle. J Invest Dermatol 33:307-316 5. Ludwig E (1967) Removal of intact hair papilla and connective tissue sheath by plucking anagen hairs. J Invest Dermatol 48: 595-597 6. MontagnaW, ParakkalPF (1974) The structure and function of skin. Academic Press, NewYork, pp 172-258 7. SkalliO, RoprazP, TrzeciakA, Benzonana G, Gillessen D, Gabbiani G (1986) A monoclonal antibody against m-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103:2787 8. Skalli O, Pelte MF, Peclet MC, Gabbiani G, Gugliotta P, Bussolati G, Ravazzola M, Orci L (1989) s-Smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles ofpericytes. J Histochem Cytochem 37:315-321
.......
rlNl~ rl
I}
,9 Springer-Verlag 1992
Identification of a cell layer containing a-smooth muscle actili in the connective tissue sheath of human anagen hair A. Urabe, M. Furumura, S. Imayama, J. Nakayama, and Y. Hori Department of Dermatology, Kyushu University, Faculty of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan Received November 12, 1991
Summary. Immunohistochemical and immunoelectron microscopy studies revealed the presence of m-smooth muscle (~-SM) actin in fibroblasts located in the connective tissue sheath (CTS) of human anagen hair follicles. Immunostaining was positive from the base of the bulb to the upper part of the lower portion of the mature anagen hair follicles. The late catagen hair follicles did not stain. Ultrastructurally, ~-SM actin was detected only in the fibroblasts located in the innermost layer of the transverse collagenous fibres. Since ~-SM actin is located in cells with contractile potential, this newly identified layer may play an important role in the morphological changes of the lower portion of the hair follicle during the hair growth cycle. Key words: H a i r - C o n n e c t i v e tissue s h e a t h - A l p h a S m o o t h m u s c l e actin - I m m u n o h i s t o c h e m i s t r y munoelectron microscopy
Im-
T h e c o n n e c t i v e tissue s h e a t h (CTS) is a c o m p o n e n t of the h a i r follicle. U l t r a s t r u c t u r a l l y , the C T S c o m p r i s e s t h r e e layers: (1) the i n n e r c o l l a g e n layer, c o m p o s e d of c o l l a g e n fibres r u n n i n g p a r a l l e l to the h a i r l o n g axis: (2) the m i d d l e c o l l a g e n layer, c o m p o s e d of t r a n s v e r s e collagen fibres a n d i n t e r m i n g l e d f i b r o b l a s t s ; a n d (3) the o u t e r c o l l a g e n layer, c o m p o s e d of c o l l a g e n fibres r u n n i n g in v a r i o u s d i r e c t i o n s a n d cellular c o n s t i t u e n t s such as fibroblasts, b l o o d vessels a n d fat cells [2, 3]. I m m u n o h i s t o chemically, several a n t i b o d i e s h a v e b e e n used to identify e p i t h e l i a l c o m p o n e n t s of h a i r follicles, b u t specific m a r kers for the C T S h a v e n o t b e e n found. A l p h a - s m o o t h m u s c l e ( ~ - S M ) actin is a d i f f e r e n t i a t i o n m a r k e r o f s m o o t h m u s c l e cells a n d is p r e s e n t in all s m o o t h m u s c l e cells a n d p e r i c y t e s [8]. W e m a d e use o f the a n t i - ~ - S M actin a n t i b o d y to o b s e r v e the presence o f this a c t i n in f i b r o b l a s t s in the C T S o f h u m a n a n a g e n hairs. P o s s i b l e f u n c t i o n s o f these cells in a s s o c i a t i o n w i t h the h a i r g r o w t h cycle a r e discussed.
Correspondence to: A. Urabe
Materials and methods
Immunohistochemistry Specimens of normal adult scalp skin were obtained from five Japanese patients (three males and two females, 53-72 years old) during surgical removal of benign turnouts. The specimens were snap-frozen in liquid nitrogen and stored at - 8 0 ~ until use. Serial frozen sections (5 gin) were placed on microscopic slides coated with poly-L-lysine (Sigma Chemicals, St. Louis, USA), airdried and fixed in cold acetone for 10min. To confirm the distribution of ~-SM actin in various stages of the hair growth cycle, two patients (males aged 13 and 51 years) with congenital melanocytic naevus were selected, from whom a H & E-stained sections showed late catagen and early anagen hair follicles. Paraffin blocks of these were recur, and 5 gm paraffin sections were de-waxed in xylene and washed in ethanol. After blocking with 2% normal horse serum, both frozen and paraffin sections were incubated overnight at 4 ~ with anti-~-SM actin antibody (Sigma Chemicals) at a dilution of 1:2000 using 0.1% bovine serum albumin (BSA) in PBS as diluent. Following incubation with biotinylated anti-mouse IgG (Immunotech, Marseille, France) at a dilulion of 1 : 40, the sections were reacted with the fluorescein-conjugated streptavidin-biotin complex (Gibco BRL, Gaithersburg, USA) at a dilulion of 1:40. Both incubations were performed for 45 min at 37 ~ The sections were examined under a Zeiss (Oberkochen, FRG) fluorescent microscope. H & E staining of serial frozen sections was carried out using conventional procedures.
Electron microscopy Specimens were fixed in 3% glutaraldehyde and post-fixed in 1% osmium tetroxide in 0.1M cacodylate buffer. The tissues were dehydrated in increasing concentrations of ethanol and embedded in Epon 812. Ultrathin tissue sections were stained with uranyl acetate and lead citrate.
Immunoelectron microscopy Specimens were fixed in periodate-lysine-paraformaldehyde,dehydrated in increasing concentrations of dimethyl formamide in water, and embedded in Lowicryl K4M at 4 ~ (Low; Waldkraiburg, FRG) as previously described [1]. Ultrathin sections, collected on nickel grids, were incubated in a drop of 1% BSA in PBS for 30 min and then incubated in anti-c~-SM actin antibody at a dilution of 1 : 100 for 1 h. After rinsing with PBS, the sections were incubated
247 in goat anti-mouseIgG labelled with 15 nm gold particles (GAMG15; Janssen, Olen, Belgium)at a dilution of 1:20 for 1 h, washed again thoroughly with water, and stained with uranyl acetate and lead citrate. For the control, the complete staining protocol was followed, but without incubation in the primary antibody. All specimens were examined under a Hitachi (Tokyo,Japan) H-7000 electron microscope.
Results
Histologically, the lower portion of the anagen hair follicle was composed of the hair, the inner root sheath, the outer root sheath, the CTS including the vitreous membrane and the dermal papilla (Figs. 1A, 2A). In late catagen, a thin epithelial cord and dermal papilla were present at the base of the follicle (Fig. 4A). Dilated capillaries were seen below the papilla. In early anagen, a newly formed hair follicle descending from the epithelial sac with club hair was observed (Fig. 5A). A high-power view of the section showed two or three layers of fibroblasts with small nuclei surrounding the vitreous membrane (Fig. 5B).
The localization of ~-SM actin was observed in vascular smooth muscle cells, pericytes and myoepithelial cells of eccrine glands in the normal human scalp skin. In mature anagen hair follicles present in all sections examined, a clear staining was seen in the CTS (Fig. 1 B) as well as arrector pili muscle. The positive reaction was observed in the bulb of the anagen hair follicles, but there was no staining in the dermal papilla (Fig. 2B). The staining faded at the upper part of the lower portion of the hair follicles and was no longer present at the arrector pili muscle (Fig. 3). No reaction was seen in the connective tissue around the hair follicles at the level of the isthmus and the infundibulum. There was no staining in the late catagen hair follicle, except for vascular smooth muscle cells and pericytes (Fig. 4B). In early anagen, a weak positive staining was observed in the CTS (Fig. 5 C). Ultrastructurally, the CTS of a mature anagen hair follicle was composed of a basal lamina and surrounding collagen tissue which was further separated into three distinct layers, the inner, intermediate and outer collagen layers, as noted by other investigators [3]. Fibroblasts located in the innermost layer of the intermediate layer are characterized bY their ramified configuration, relatively dense cytoplasm filled with fine filaments and a few cytoplasmic organellas (Fig. 6). Immunoelectron microscopy revealed that anti-~-SM actin antibody labelled the cytoplasm of these fibroblasts (Fig. 7) but not the fibroblasts located in the outer area of the intermediate layer and in the outer collagen layer. Pericytes surrounding the small vessels in the CTS were also labelled with this antibody. Discussion
Fig. 1A. The lower portion of mature anagen hair follicle (H & E, 90). B The CTS stained with anti-c~-SMactin antibody (100) Fig. 2A. The bulb of mature anagen hair follicle (H & E, 90). B Note the lack of staining of the dermal papilla (100)
We obtained evidence that ~-SM actin is present in fibroblasts located in the innermost layer of the transversely running collagen fibres of the CTS. Since ~-SM actin has previously been found only in cells with contractile function [7] and the presence of this actin was confined to the ower portion of the hair follicles, it seems reasonable to speculate that these cells play an important role in the hair growth cycle involving dynamic kinetics of the lower portion of the hair follicles. The hair cycle is composed of three different stages; i.e. anagen (proliferative phase), catagen (subsiding growth) and telogen (resting phase). At the beginning of catagen, the club-shaped, keratinized proximal shaft is pushed upwards by a column of epithelial cells, and subsequently the dermal papilla moves upwards as the epithelial column shortens [4]. Montagna and Parakkal [6] found that the collagen fibres in the CTS were broken down during the process by the tightly packed layer of macrophages, and they speculated that macrophages participated in the degenerative changes of the CTS. However, Ito and Sato [3] recently demonstrated that macrophages did not play a specific role in the dynamic changes observed in catagen, and they speculated that the CTS around the lower end of the epithelial cord was digested by enzymes produced by the epithelial cells. We
248
Fig. 3. The upper part of the lower portion of the mature anagen hair. A positive staining of the CTS (arrowhead) discontinuouswith that of the arrector pill muscle (arrow) (50) Fig. 4A. The late catagen hair follicle in paraffin section (H & E, 90). B Note the lack of staining around the hair follicle, except for vascular smooth muscle cells and pericytes (100) Fig. 5A. The early anagen hair follicle in paraffin section. The telogen club hair (T) was present within its epithelia1 coating (H & E, 45). B High-power view of the early anagen hair follicle (H & E, 180). C The CTS of the early anagen hair follicle was weakly stained with the antibody (200)
Fig. 6. Electron micrograph of the CTS of the mature anagen hair. The inner collagen layer (a), located next to the basal lamina (arrowheads), was composed of collagen fibres running parallel to the long axis of the hair. The intermediate layer (b) was composed of collagen fibres running transversely against the hair axis and fibroblasts (*). The outer layer (c) consisted of irregularly arranged
collagen fibres intermingled with fibroblasts and small blood vessels (bar, 1 ~tm) Fig. 7. Electron micrograph of the fibrobIasts immunolabelled with anti-c~-SM actin antibody. Gold particles are distributed in the cytoplasm (bar, 0.1 p,m)
249 propose that retraction of the epithelial cord and the basal lamina m a y be caused by the contractile function of the fibroblasts present in the innermost layer of the CTS. Ultrastructurally, both the basal lamina and the inner collagen layer of the CTS present outside the basal lamina showed a wavy appearance during catagen [3]. This morphological change supports the notion of a contractile function of these cells present around the inner collagen layer. The expression of ~-SM actin by fibroblasts in the CTS m a y be useful for evaluating histogenesis of tumours considered to be of hair follicle origin. This anti-~-SM actin antibody m a y also be of use for analysing the origin of cultured hair follicle cells, since plucked anagen hairs sometimes contain the CTS [5]. The newly identified thin layer composed of fibroblasts stained with anti-~-SM actin antibody described here probably has a characteristic function and studies to identify this putative function are underway.
Acknowledgement. The authors thank M. Ohara for critical comments on the manuscript.
References 1. Altman LG, Schneider BG, Papermaster DS (1984) Rapid embedding of tisses in Lowicryl K4M for immunoelectron microscopy. J Histochem Cytochem 32:1217-1223 2. Hashimoto K (1981) The environment of the hair follicles. In: Orfanos CE, Montagna W, Stuttgen G (eds) Hair research. Springer, Berlin, Heidelberg, New York, pp 172-182 3. Ito M, Sato Y (1990) Dynamic ultrastructural changes of the connective tissue sheath of human hair follicles during hair cycle. Arch Dermatol Res 282:434-441 4. Kligman AM (1959) The human hair cycle. J Invest Dermatol 33:307-316 5. Ludwig E (1967) Removal of intact hair papilla and connective tissue sheath by plucking anagen hairs. J Invest Dermatol 48: 595-597 6. MontagnaW, ParakkalPF (1974) The structure and function of skin. Academic Press, NewYork, pp 172-258 7. SkalliO, RoprazP, TrzeciakA, Benzonana G, Gillessen D, Gabbiani G (1986) A monoclonal antibody against m-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103:2787 8. Skalli O, Pelte MF, Peclet MC, Gabbiani G, Gugliotta P, Bussolati G, Ravazzola M, Orci L (1989) s-Smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles ofpericytes. J Histochem Cytochem 37:315-321
