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Angiogenesis in pathological and surgical scars
Angiogenesis in Pathological and Surgical Scars TREVOR W. BEER, MRCPATH, HEATHER C. BALDWIN, FRCOPHTHI JONATHON R. GODDARD, BSc, PATRICK J. GALLAGHER, MD AND DENNIS H. WRIGHT, MD We identified immmmhistochemically vessels within cutaneous scars to test the hypothesis that they are qualitatively and quantitatively different in pathological and normally healing scars. The vascular density within and. adjacent to 58 cutaneous scars was measured on histological sections stained for factor VIII-related antigen. Four scar types were studied; surgical scars (n = 25), hypertrophic scars (n = 10), keloid (n = 12), and periocular scars (n = 11). Statistical analyses were performed to test the hypothesis that vessel numbers were different in the varying types of scar. Results were also compared with mast cell counts previously derived from the same tissues. A significant reduction was found in the vascular density in
keloids compared with surgical and hypertrophic scars (P < .05). No correlation was found between the vascular density and the number of mast ceils in the lesions. We have shown quantitatively that keloids have a reduced vascular component compared with hypertrophic scars and normally healing surgical scars at periocular and systemic sites. The formation of keloid scars may be related to their reduced level of vascularization mediated through tissue hypoxia. HUM PaTHOL 29:1273-1278. Copyright © 1998 by W.B. Saunders Company Key words: angiogenesis, keloid, hypertrophic scar, cicatrix. Abbreviations: TGF-[~, transforming growth factor beta,
P a t h o l o g i c a l scar r e a c t i o n s such as k e l o i d s a n d h y p e r t r o p h i c scars m a y result i n s i g n i f i c a n t f u n c t i o n a l a n d psychological morbidity. T h e s e processes are the result of excess fibrous tissue d e p o s i t i o n d u r i n g c u t a n e : ous h e a l i n g , a n d a l t h o u g h m a n y p r e d i s p o s i n g factors a n d associations are r e c o g n i z e d , the cellular m e c h a n i s m s involved are n o t fully established. I n this Study we i n c l u d e d tissue f r o m p e r i o c u l a r scars b e c a u s e keloids are rare lesions o f the eyelids b u t are f r e q u e n t l y sited elsewhere o n the h e a d a n d neck. >a It is i m p o r t a n t clinically to b e able to d i s t i n g u i s h keloid a n d h y p e r t r o p h i c scars, b u t this is n o t always possible with c u r r e n t m e t h o d s . 4 A n g i o g e n e s i s plays a crucial role i n skin h e a l i n g , a n d t h e r e are r e p o r t s of m i c r o v a s c u l a r a b n o r malities i n p a t h o l o g i c a l scars, s-6Vessels w i t h i n scars were s t u d i e d a n d q u a n t i f i e d by i m m u n o h ! s t o c h e m i s t r y u s i n g a n a n t i b o d y to facto r V I I I - r e l a t e d a n t i g e n to investigate the hypothesis t h a t vessels are qualitatively a n d q u a n t i t a tively d i f f e r e n t i n p a t h o l o g i c a l a n d n o r m a l l y h e a l i n g scars at p e r i o c u l a r a n d o t h e r sites.
brief, keloids are well circumscribed, raised lesions that may extend beyond the site of ofiginal skin damage. By contrast, hypertrophic scars remain confined to the site of skin injury and are less likely to recur. Histologically, keloids are characterized by nodular fibroplasia with hypocellular dermal collagenization. Hypertrophic scars are more cellular lesions and lack t h e hyaline "glassy" collagen associated with keloids, Healing scar tissue "surgical scars" (n = 25) was obtained from wider surgical excisions after primary malignant melanoma resection. Only cases in which there was no evidence of recurrent/residual melanoma were used. Cases of periocular healing scars, "periocular scars" (n = 11), were derived from nonpathological scar tissue excised after previous surgery at the same site. This was related to revision procedures for ptosis and dermatochalasis. The ages of the surgical scar subjects were between 24 and 86 years (median, 48.0), with 10 women and 15 men. All lesions were sited on the leg, trunk, or face and were of 8 to 83 days' duration. Hypertrophic scar patients were aged from 14 to 66 years (median, 29.0), with four females and six males. The scars were located on the head, neck, upper limb, and in one case, the foot. Data for the scar duration was available in only four cases; these were between 12 and 22 months old. Keloid scars were located on the head, neck, upper trunk, and upper limb in subjects 11 to 74 years of age (median, 34.5). Five patients were male and seven female. The duration of tile keloid scars was known for only six patients and was between 18 and 96 months. The periocular scars were derived from the lower or upper eyelid in patients between 28 and 80 years (median, 57.0), one male and eight females. One scar was known to be 49 days old, but data were not available for the remaining cases. Samples from the left and fight eyelids of one subject were used (cases 5 and 6). Wound infection was not reported in any of the scars studied. ' .
METHODS Paraffin-embedded blocks were retrieved from the archives of The Department of Pathology at Southampton University Hospitals Trust. All cases were reviewed on hematoxylin and eosin-stained sections and catagorized as hypertrophic scars (n = 10) or keloids (n = 12) by their clinical and histological features according to recognized criteria. 4,8,9 In
From the Departments of Histopathology, Southampton Eye Unit, and Department of Medical Statisticsand Computing, Southampton University Trust Hospital, Southampton, United Kingdom. Accepted for publication May 15, 1998. Supported by a Grant from the South and West Regional Health Authority, Bristol, UIC Address correspondence and reprint requests to T.W. Beer, Department of Histopathology, RoyalHospital Haslar, Gosport, Hampshire, PO12 2AA. Copyright © 1998by W.B. SaundersCompany 0046-8177/98/2911-001558.00/0
Immunohistochemistry Five-micron paraffin block-derived sections from each case were stained immunohistochemically for factor VIlIrelated antigen using the streptavidin biotin method. Sections were pretreated with 0.1% trypsin. Rabbit polyclonal antibody to factor VIII (Dako UK Ltd, UK) was used at a dilution of I in 500 for 30 minutes at room temperature. Appropriate positive
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c o n t r o l s were p e r f o r m e d , a n d in t h e negative c o n t r o l s t h e p r i m a r y a n t i s e r u m was r e p l a c e d with Tris-buffered saline.
Vessel Counting Scar tissue was identified o n t h e h e m a m x y l i n a n d e o s i n s t a i n e d sections a n d t h e c o r r e s p o n d i n g area i d e n t i f i e d o n t h e i m m u n o h i s t o c h e m i c a l p r e p a r a t i o n s . A Chalkley eyepiece graticule (Graticules U K L i m i t e d , UK) was s u p e r i m p o s e d over t h e tissue, a n d t h e n u m b e r o f p o i n t s falling o n cells clearly l a b e l e d i m m u n o h i s t o c h e m i c a l l y , or within l u m i n a d e l i n e a t e d by factor VIII-positive cells was recorded. C o u n t i n g was p e r f o r m e d at a m a g n i f i c a t i o n o f 250 times using a Lietz Dialux 20 m i c r o s c o p e (Leica U K L i m i t e d . UK) with a field a r e a o f 0.628 mm2. T e n s e q u e n t i a l fields were analyzed f r o m t h e superficial to d e e p area o f t h e scar, a n d a vessel score was derived, r e p r e s e n t i n g t h e n u m b e r o f hits o n e n d o t h e l i a l cells or vessel l u m i n a p e r s q u a r e millimeter. A similar vessel score was r e c o r d e d in 10 m i c r o s c o p i c fields f r o m histologically n o r m a l d e r m i s s u r r o u n d i n g t h e scars, u p to a d e p t h o f 0.4 m m f r o m t h e overlying e p i t h e l i u m .
Statistical Analyses One-way analysis of v a r i a n c e with s u b s e q u e n t pairvase c o m p a r i s o n s were u s e d to c o m p a r e t h e m e a n vessel scores b e t w e e n t h e d i f f e r e n t scar groups. This was p e r f o r m e d for
TABLE 1.
c o u n t s w i t h i n the scar tissue a n d in the s u r r o u n d i n g dermis. P e a r s o n c o r r e l a t i o n coefficients o f vessel score a n d m a s t cell n u m b e r s were calculated a n d assessed for significance w i t h i n e a c h g r o u p o f scars. Vessel scores were also c o m p a r e d b e t w e e n m a t u r e surgical scars o f 35 o r m o r e days d u r a t i o n a n d r e c e n t scars p r e s e n t for less t h a n 35 days, u s i n g a two-sample t-test.
RESULTS In all cases, factor VIII clearly showed e n d o t h e l i a l cells in the scar a n d s u r r o u n d i n g d e r m i s a n d readily facilitated vessel quantification (Table 1, Fig 1). T h e m e a n vessel score in keloids (Table 2) was significantly less than in either surgical or h y p e r t r o p h i c scars (P < .05). T h e p e r i o c u l a r scars s h o w e d a m e a n vessel score b e t w e e n that o f keloids a n d h y p e r t r o p h i c or surgical scars, but the difference was not statistically significant (Fig 2). In the d e r m i s s u r r o u n d i n g the scars, n o significant difference was f o u n d in vessel score b e t w e e n the scar types. Statistical c o m p a r i s o n with d e r m i s s u r r o u n d i n g p e r i o c u l a r scars was n o t possible b e c a u s e of the small a m o u n t of tissue available in s o m e cases. C o m p a r i n g r e c e n t surgical scars (less t h a n 35 days duration, n = 13) a n d older scars (35 or m o r e days
Actual VesselScoresfor al! Scar Types
Vessel Score*
Surgical scars
Periocular scars
Vessel Score*
Lesion
Dermis
Scar Duration (days)
0.96 2.55 2.23 2,55 1,59 0,96 1,75 0.48 1.11 1.11 3.98 2.O7 3.18 1.59 1.75 1.59 2.23 2.07 1.11 2.23 1.59 1.27 1.75 1.75 1.43 1.59 1.11 0.64 0.64 2.23 2.23 1.27 1.91 0.80 1.75 2.39
1.27 1.75 1.27 0.48 1.91 1.43 2.55 1.27 0132 0.64 8.76 1.75 0.96 0,80 0.48 1,59 2.87 1.59 0.32 0.96 2.07 1.59 2.39 1.75 1.43 ND ND ND ND ND ND 1.59 ND ND 2.39 ND
8 13 21 23 26 26 28 29 30 32 32 32 33 35 35 36 36 37 37 39 46 49 49 5O 83 49 ND ND ND ND ND ND ND ND ND ND
Scar Duration Lesion
Dermis
(too)
Hypertrophic scars
1.59 2.71 3.50 0.96 1.11 1.27 2.87 1.43 1.11 2.71
0.96 1.91 1.11 1.75 3.03 2.07 1.il 3.03 1.43 1.27
12 12 12 22 ND ND ND ND ND ND
Keloid scars
0.16 2.55 0.64 O.8O 0.80 0.80 1.27 !. 11 0.48 0.96 0.64 1.27
1.27 3.34 2.23 2.07 3.03 0.64 4.94 2.39 3.34 1.27 3.66 1.43
18 22 26 30 60 96 ND ND ND ND ND ND
Abbreviations: ND, no data available. *Vessel score = number of Chalkley point "hits" with endothelial ceils or vessel lumina per square millimeter.
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FIGURE 1. Hypertrophic scar showing a dense proliferation of fine vessels. (Immunohistochemical stain for factor VIII-related antigen; original magnification x 160.)
d u r a t i o n , n = 12), n o s i g n i f i c a n t d i f f e r e n c e in vessel scores was s e e n f o r e i t h e r l e s i o n a l o r d e r m a l tissue. However, r e c e n t scars d i d s h o w n e a r l y twice as m u c h variability in vessel s c o r e w h e n s t a n d a r d d e v i a t i o n s w e r e c o m p a r e d . T h e r e were insufficient d a t a to allow statistically valid c o m p a r i s o n o f vessel s c o r e a n d scar d u r a t i o n f o r t h e o t h e r scar types. W i t h i n s o m e o f t h e cases o f k e l o i d s c a r r i n g , t h e r e was a subjective r e d u c t i o n in vessel l u m e n a l d i a m e t e r . This a p p e a r e d to b e r e l a t e d to p r o m i n e n t e n d o t h e l i a l cells p r o j e c t i n g i n t o t h e l u m e n (Fig 3). T h e f i n d i n g was o f a focal n a t u r e , a n d n o f i b r i n d e p o s i t i o n o r t h r o m b u s were i d e n t i f i e d . I n c o n t r a s t , in the dermis immediately surrounding some of the nodules o f k e l o i d a l tissue, vessels a p p e a r e d d i l a t e d with a less c o n s p i c u o u s e n d o t h e l i a l lining. I n s o m e o f t h e h y p e r t r o p h i c scar cases, a s i m i l a r p a t t e r n o f fine vessels with p a r t l y o b s t r u c t e d l u m i n a was also n o t e d , a n d s u r r o u n d i n g v a s c u l a r d i l a t i o n was also s e e n in s o m e cases. S u r g i c a l scars g e n e r a l l y s h o w e d m o r e widely p a t e n t vessels t h a n k e l o i d o r h y p e r t r o p h i c scars, b u t in s o m e e x a m p l e s vessels with p r o m i n e n t e n d o t h e l i a l cells a n d o n l y a tiny l u m e n were i d e n t i f i e d . T h e surgical scars l a c k e d t h e p e r i p h e r a l v a s c u l a r d i l a t i o n s e e n in k e l o i d a n d h y p e r t r o p h i c scars. Mast cell n u m b e r s in a n d a r o u n d e a c h o f t h e s e scars h a d b e e n m e a s u r e d in a TABLE 2.
p r e v i o u s study. :° T h e r e was n o r e l a t i o n s h i p b e t w e e n t h e n u m b e r s o f m a s t cells a n d vessel s c o r e f o r a n y scar type. DISCUSSION This s t u d y p r o v i d e s q u a n t i t a t i v e e v i d e n c e t h a t kel o i d scars have a r e d u c e d vascular d e n s i t y c o m p a r e d with h y p e r t r o p h i c scars a n d surgical scars at p e r i o c u l a r a n d systemic sites. I n a d d i t i o n to a r e d u c e d vascular density, s o m e k e l o i d s s h o w e d subjective p a r t i a l occlus i o n o f vascular l u m i n a by p r o m i n e n t e n d o t h e l i a l cells. T h i s was s e e n in s o m e cases o f m a n y m o n t h s ' d u r a t i o n , suggesting that they do not merely represent new c a p i l l a r i e s j u s t b e g i n n i n g to f o r m h o l l o w l u m i n a . Simi-
3.5
3.0
~. 0
2.5
g 2.0
g
1.5
M e a n Vessel Score of the Four Scar Types
Mean Vessel Score* (Standard Deviation)
Keloid scars Surgical scars Hypertrophic scars Periocnlar scars
1.0
Lesion
Dermis
Ratio Mean Vessel Score Lesion: Dermis
0.96 (0.6) 1.80 (0.75) 1.93 (0.92) 1.51 (0.65)
2.47 (1.24) 1.69 (1.62) 1.77 (0.76) 1.99 (0.57)
0.39 1.07 1.09 0.76
.5 ¸
0.0. 11 Periocular
1~2 Hypertrophic
12 Keloid
25 Surgical
Scar type
*Vesselscore = number of Chalkley point "hits" with endothelial cells or vessel lumina per square millimeter.
FIGURE 2. Vessel scores of the four scar types with 95% confidence intervals.
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FIGURE3, Keloid scar showing a low vascular complement. Note the presence of a number of fine vessels with largely o c c l u d e d lumina (arrows). (Immunohistochemical stain for factor Viii-related antigen; original magnification x 160.)
larly narrowed vessels were also n o t e d in some hypertrophic and surgical scars. O t h e r groups have stated that hypertrophic or keloid scars have an increased vascular c o m p o n e n t compared with healing scars, 5-7 but these suggestions were based on qualitative studies with small numbers of cases. T h e concept of keloid hypervascularity has b e c o m e perpetuated in the literature 3,8 with suggestions that angiogenic factors from macrophages may enhance the growth of keloids. 11 Mast cells may also have an angiogenic action, le but our previous findings have questioned the role of mast cells in stimulating keloid development. 1° Microvascular occlusion in hypertrophic scars and keloids has been well documented, 5,7,8,1s although this change is not confined to pathological scars. One of the key studies in this area f o u n d that occluded vessels were present in very similar numbers in keloids, mature scars, and normal skin. 5 Recently, a correlation between myocardial fibrosis and capillary Reduced angiogenesis
\
Wound tension / infection
FIGURE4. Hypoxia is of crucial importance in the development of keloid scars.
luminal narrowing has b e e n d o c u m e n t e d in hypertrophic hearts. 14 Vascular dilation at the peripheral margins of the lesions was noted only for hypertrophic scars and keloids. Vessel numbers in histologically normal dermis surrounding the scars were similar for all scar types. Keloid and hypertrophic scars are known to be hypoxic, 7 and we postulate that in keloids hypoxia is mediated through their reduced vascularity c o m b i n e d with microvascular occlusion. O t h e r factors are clearly also important, but hypoxia is likely to play a crucial role (Fig 4). Keloids occurring at sites of skin tension and in wounds that have been infected or u n d e r tension are well documented, and these circumstances may cause tissue hypoxia. Although hypoxia appears important in the development and maintenance of keloids, severe or "critical hypoxia" may cause their involution. Prolonged external pressure applied to keloids is known to reduce scar severity, 15A6 and this could be mediated
Abnormal endothelial cells obstructing vessel lumen
E HYPOXIA
~
KELOID FORMATION
I
Sites of skin tension
Irradiation External pressure
Excess collagen deposition by fibroblasts Critical hypoxia
1 1
Fibroblast death
Keloid involution
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through vascular compression resulting in fibroblast death from ischemia. Irradiation of keloids may also reduce their severity, 17 possibly through ischaemia produced by microvascular damage. Excess collagen deposition by fibroblasts in keloids 6,7'n'18'19 inhibits oxygen diffusion, promoting tissue hypoxia and further collagen production. Transforming growth factor-beta (TGF-[3) promotes increased collagen production by fibroblasts and is increased in keloids and hypertrophic scars. 2°,21 The crucial role of TGF-[3 has b e e n demonstrated by neutralizing its effects with specific antibodies, which leads to decreased collagen production and reduced scarring. 18,21-23 Scarless healing in fetal tissue is characterized by an absence of TGF-[~ at the wound site. 24 The role of TGF-[3 in angiogenesis is unclear 25 and warrants further investigation. The reduced vascularity of keloids may also promote collagen deposition through a lack of the inhibitory action of factor XIIIa-positive dermal dendrocytes. Keloids are known to have a low c o m p l e m e n t of these cells, 2e-28 which inhibit collagen synthesis 28-~° and are closely associated with vessels. 3~,~2Factor XIIIa has also been localized to endothelial cells by electron microscopy? 3 Mast cells may promote collagen deposition 11,18 and angiogenesis 12,~4,3~ mediated by heparin, but we could not confirm a relation between mast cells and angiogenesis in these types of scar. In a previous study, 1° we have f o u n d no difference in mast cell numbers between keloids, hypertrophic scars, and surgical scars in nonperiocular sites. Periocular tissue scars were, however, f o u n d to have increased numbers of mast cells c o m p a r e d with all other scar types.
CONCLUSION Keloids show a quantitative reduction in vessels compared with hypertrophic and surgical scars at periocular and other sites. This helps to explain their hypoxic nature, especially because there may be some occlusion of the scant vessels present. Vascular quantification may help in making the important distinction between hypertrophic and keloid scars, ~ and this m e t h o d goes some way to realizing the goal of a laboratory test to separate the two as envisaged by Norris. 37 Keloid scar formation is associated with tissue hypoxia, which is partly mediated by reduced vascularization.
Acknowledgment. T h e a u t h o r s t h a n k R. M a n n e r s for s u p p l y i n g m a t e r i a l f r o m t h e p e r i o c u l a r scars a n d to L. West for t e c h n i c a l assistance. REFERENCES I. Chrisman BB: Blepharoplasty and browlift with surgical variations in non-white patients.J Dermatol Surg Oncol 12:58-66, 1986 2. Offret H, Quillard J: Nodule cheloidien du bord libre de la paupiere.J Fr Ophthalmo116:552-553, 1993 3. Rockwell WB, Cohen IK, Ehrlich HP: Keloids and hypertrophic scars: A comprehensive review. Plast Reconstr Surg 84:82%837, 1989
4. McGrouther DA: Hypertrophic or keloid scars? Eye 8:200-203, 1994 5. Kischer CW, Thies AC, Chvapil M: Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids. HuM PATHOL 13:819-824, 1982 6. Thomas DW, Hopkinson I, Harding KG, et ah The pathogenesis of hypertrophic/keloid scarring. Int J Oral MaxiIlofac Surg 23:232-236, 1994 7. Kischer CW: The microvessels in hypertrophic scars, keloids and related lesions: A review. J Submicrosc Cytol Pathol 24:281-296, 1992 8. Ehrlich HE Desmouliere A, Diegelmann RF, et ah Morphological and immunochemical differences between keloid and hypertrophic scar. AmJ Patho1145:105-113, 1994 9. McKee PH (ed): Pathology of the Skin with Clinical Correlations (ed 2). London, England, Mosby-Wolfe, 1996, pp 16.8-16.9 10. Beer TW, Baldwin H, West L, et al: Mast cells at pathological and surgical scar sites. BrJ Ophthalmo182:691-694, 1998 11. Smith CJ, Smith JC, Finn MC: The possible role of mast cells (allergy) in the production of keloid and hypertrophic scarring. J Burn Care Rehabil 8:126-131, 1987 12. Meininger CJ, Zetter BR: Mast Cells andAngiogenesis. Semin Cancer Biol 3:73-79, 1992 13. Lametschwandtner A, Staindl O: The vascular architecture of the keloid: A scanning electron microscope study on vascular corrosion casts. HNO 38:202-207, 1990 14. Takemura G, Takatsu Y, Fujiwara H: Luminal narrowing of capillaries in human hypertrophic hearts: An ultrastructural morphometrical study using endomyocardial biopsy specimens. Heart 79:7885, 1998 15. Zuber TJ, DeWitt DE: Earlobe keloids. Am Faro Physician 49:1835-1841, 1994 16. Murray JC: Scars and keloids. Dermatol Clin 11:69%708, 1993 17. NorrisJE: Superficial x-ray therapy in keloid management: A retrospective study of 24 cases and literature review. Plast Reconstr Surg 95:1051-1055, 1995 18. Suzawa H, Kikuchi S, Arai N, et al: The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts.JpnJ Pharmaco160:91-96, 1992 19. Riaz Y, Cook HT, Wangoo A, et al: Type 1 procollagen as a marker of severity of scarring after sternotomy: Effects of topical corticosteroids. J Clin Pathol 47:892-899, 1994 20. Zhang K, Garner W, Cohen L, et al: Increased types I and III collagen and transforming growth factor-beta 1 mRNA and protein in hypertrophic burn scar.J Invest Dermato1104:750-754, 1995 21. Border WA, Nobe NA: Transforming growth factor [3 in tissue fibrosis. N EnglJ Med 331:1286-1292, 1994 22. Shah M, Foreman DM, Ferguson MWJ: Neutralisation of TGF-beta 1 and TGF-beta2 or exogenous addition of TGF-beta3 to cutaneous rat wounds reduces scarring. J Cell Sci 108:985-1002, 1995 23. Ferguson MWJ: Skin wound healing: Transforming growth factor beta antagonists decrease scarring and improve quality. J Interferon Res 14:303-304, 1994 24. Nath RK, LaRegina M, Markham H, et ah The expression of transforming growth factor type beta in fetal and adult rabbit skin wounds.J Pediatr Surgy 29:416-421, 1994 25. Levine JH, Moses HL, Gold LI, et al: Spatial and temporal patterns ofimmunoreactive transforming growth factor 131,[32,and [33 during excisional wound repair. AmJ Patho1143:368-380, 1993 26. Altman DA, Nickoloff BJ, Fivenson DP: Differential expression of factor XIIIa and CD34 in cutaneous mesenchymal tumours. J Cutan Pathol 20:154-158, 1993 27. Cerio R, SpaullJ, Jones EW: Histiocytoma cuffs: A tumour of dermal dendrocytes (dermal dendrocytoma). BrJ Dermatol 120:197206, 1989 28. Pierard GE, Arrese-EstradaJ, Pierard-Franchimont C, et al: Is there a link between dendrocytes, fibrosis and sclerosis? Dermatologica 181:264-265, 1990 29. Pierard-Franchimont C, Arrese JE, Nikkels AF, et al: Factor XIIIa-positive dendrocytes and proliferative activity of cutaneous cancers. Virchows Arch A Pathol Anat Histopathol 429:43-48, 1996
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30. Hoyo E, Kanitakis J, Schmitt D: The dermal dendrocyte. Pathol Bio141:613-618, 1993 31. Suvarna SK, Cotton DWK: Dermal dendrocytes and other factor XIIIA-positive cells.J Pathol 171:251-252, 1993 32. Penneys NS: Factor XIII expression in the skin: Observations and a hypothesis.J Am Acad Dermatol 22:484488, 1990 33. Schaumburg-Lever G, Gehring B, Kaiserling E: Ultrastructural localisation of factor XIIIa. J Cutan Pathol 21:129-134, 1994
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34. Trabucchi E, Radaelli E, Marazzi M, et al: The role of l~last cells in wound healing. IntJ Tissue Reac X:367-372, 1988 35. Arbiser JL: Angiogenesis and the skin: A primer. J Am Acad Dermato134:486-497, 1996 36. Brody GS: Keloids and hypertrophic scars. Plast Reconstr Surg 86:804, 1990 37. NorrisJE: Hypertrophic scar and keloid [letter]. Plast Reconstr Surg 88:372-373, 1991
keloids compared with surgical and hypertrophic scars (P < .05). No correlation was found between the vascular density and the number of mast ceils in the lesions. We have shown quantitatively that keloids have a reduced vascular component compared with hypertrophic scars and normally healing surgical scars at periocular and systemic sites. The formation of keloid scars may be related to their reduced level of vascularization mediated through tissue hypoxia. HUM PaTHOL 29:1273-1278. Copyright © 1998 by W.B. Saunders Company Key words: angiogenesis, keloid, hypertrophic scar, cicatrix. Abbreviations: TGF-[~, transforming growth factor beta,
P a t h o l o g i c a l scar r e a c t i o n s such as k e l o i d s a n d h y p e r t r o p h i c scars m a y result i n s i g n i f i c a n t f u n c t i o n a l a n d psychological morbidity. T h e s e processes are the result of excess fibrous tissue d e p o s i t i o n d u r i n g c u t a n e : ous h e a l i n g , a n d a l t h o u g h m a n y p r e d i s p o s i n g factors a n d associations are r e c o g n i z e d , the cellular m e c h a n i s m s involved are n o t fully established. I n this Study we i n c l u d e d tissue f r o m p e r i o c u l a r scars b e c a u s e keloids are rare lesions o f the eyelids b u t are f r e q u e n t l y sited elsewhere o n the h e a d a n d neck. >a It is i m p o r t a n t clinically to b e able to d i s t i n g u i s h keloid a n d h y p e r t r o p h i c scars, b u t this is n o t always possible with c u r r e n t m e t h o d s . 4 A n g i o g e n e s i s plays a crucial role i n skin h e a l i n g , a n d t h e r e are r e p o r t s of m i c r o v a s c u l a r a b n o r malities i n p a t h o l o g i c a l scars, s-6Vessels w i t h i n scars were s t u d i e d a n d q u a n t i f i e d by i m m u n o h ! s t o c h e m i s t r y u s i n g a n a n t i b o d y to facto r V I I I - r e l a t e d a n t i g e n to investigate the hypothesis t h a t vessels are qualitatively a n d q u a n t i t a tively d i f f e r e n t i n p a t h o l o g i c a l a n d n o r m a l l y h e a l i n g scars at p e r i o c u l a r a n d o t h e r sites.
brief, keloids are well circumscribed, raised lesions that may extend beyond the site of ofiginal skin damage. By contrast, hypertrophic scars remain confined to the site of skin injury and are less likely to recur. Histologically, keloids are characterized by nodular fibroplasia with hypocellular dermal collagenization. Hypertrophic scars are more cellular lesions and lack t h e hyaline "glassy" collagen associated with keloids, Healing scar tissue "surgical scars" (n = 25) was obtained from wider surgical excisions after primary malignant melanoma resection. Only cases in which there was no evidence of recurrent/residual melanoma were used. Cases of periocular healing scars, "periocular scars" (n = 11), were derived from nonpathological scar tissue excised after previous surgery at the same site. This was related to revision procedures for ptosis and dermatochalasis. The ages of the surgical scar subjects were between 24 and 86 years (median, 48.0), with 10 women and 15 men. All lesions were sited on the leg, trunk, or face and were of 8 to 83 days' duration. Hypertrophic scar patients were aged from 14 to 66 years (median, 29.0), with four females and six males. The scars were located on the head, neck, upper limb, and in one case, the foot. Data for the scar duration was available in only four cases; these were between 12 and 22 months old. Keloid scars were located on the head, neck, upper trunk, and upper limb in subjects 11 to 74 years of age (median, 34.5). Five patients were male and seven female. The duration of tile keloid scars was known for only six patients and was between 18 and 96 months. The periocular scars were derived from the lower or upper eyelid in patients between 28 and 80 years (median, 57.0), one male and eight females. One scar was known to be 49 days old, but data were not available for the remaining cases. Samples from the left and fight eyelids of one subject were used (cases 5 and 6). Wound infection was not reported in any of the scars studied. ' .
METHODS Paraffin-embedded blocks were retrieved from the archives of The Department of Pathology at Southampton University Hospitals Trust. All cases were reviewed on hematoxylin and eosin-stained sections and catagorized as hypertrophic scars (n = 10) or keloids (n = 12) by their clinical and histological features according to recognized criteria. 4,8,9 In
From the Departments of Histopathology, Southampton Eye Unit, and Department of Medical Statisticsand Computing, Southampton University Trust Hospital, Southampton, United Kingdom. Accepted for publication May 15, 1998. Supported by a Grant from the South and West Regional Health Authority, Bristol, UIC Address correspondence and reprint requests to T.W. Beer, Department of Histopathology, RoyalHospital Haslar, Gosport, Hampshire, PO12 2AA. Copyright © 1998by W.B. SaundersCompany 0046-8177/98/2911-001558.00/0
Immunohistochemistry Five-micron paraffin block-derived sections from each case were stained immunohistochemically for factor VIlIrelated antigen using the streptavidin biotin method. Sections were pretreated with 0.1% trypsin. Rabbit polyclonal antibody to factor VIII (Dako UK Ltd, UK) was used at a dilution of I in 500 for 30 minutes at room temperature. Appropriate positive
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c o n t r o l s were p e r f o r m e d , a n d in t h e negative c o n t r o l s t h e p r i m a r y a n t i s e r u m was r e p l a c e d with Tris-buffered saline.
Vessel Counting Scar tissue was identified o n t h e h e m a m x y l i n a n d e o s i n s t a i n e d sections a n d t h e c o r r e s p o n d i n g area i d e n t i f i e d o n t h e i m m u n o h i s t o c h e m i c a l p r e p a r a t i o n s . A Chalkley eyepiece graticule (Graticules U K L i m i t e d , UK) was s u p e r i m p o s e d over t h e tissue, a n d t h e n u m b e r o f p o i n t s falling o n cells clearly l a b e l e d i m m u n o h i s t o c h e m i c a l l y , or within l u m i n a d e l i n e a t e d by factor VIII-positive cells was recorded. C o u n t i n g was p e r f o r m e d at a m a g n i f i c a t i o n o f 250 times using a Lietz Dialux 20 m i c r o s c o p e (Leica U K L i m i t e d . UK) with a field a r e a o f 0.628 mm2. T e n s e q u e n t i a l fields were analyzed f r o m t h e superficial to d e e p area o f t h e scar, a n d a vessel score was derived, r e p r e s e n t i n g t h e n u m b e r o f hits o n e n d o t h e l i a l cells or vessel l u m i n a p e r s q u a r e millimeter. A similar vessel score was r e c o r d e d in 10 m i c r o s c o p i c fields f r o m histologically n o r m a l d e r m i s s u r r o u n d i n g t h e scars, u p to a d e p t h o f 0.4 m m f r o m t h e overlying e p i t h e l i u m .
Statistical Analyses One-way analysis of v a r i a n c e with s u b s e q u e n t pairvase c o m p a r i s o n s were u s e d to c o m p a r e t h e m e a n vessel scores b e t w e e n t h e d i f f e r e n t scar groups. This was p e r f o r m e d for
TABLE 1.
c o u n t s w i t h i n the scar tissue a n d in the s u r r o u n d i n g dermis. P e a r s o n c o r r e l a t i o n coefficients o f vessel score a n d m a s t cell n u m b e r s were calculated a n d assessed for significance w i t h i n e a c h g r o u p o f scars. Vessel scores were also c o m p a r e d b e t w e e n m a t u r e surgical scars o f 35 o r m o r e days d u r a t i o n a n d r e c e n t scars p r e s e n t for less t h a n 35 days, u s i n g a two-sample t-test.
RESULTS In all cases, factor VIII clearly showed e n d o t h e l i a l cells in the scar a n d s u r r o u n d i n g d e r m i s a n d readily facilitated vessel quantification (Table 1, Fig 1). T h e m e a n vessel score in keloids (Table 2) was significantly less than in either surgical or h y p e r t r o p h i c scars (P < .05). T h e p e r i o c u l a r scars s h o w e d a m e a n vessel score b e t w e e n that o f keloids a n d h y p e r t r o p h i c or surgical scars, but the difference was not statistically significant (Fig 2). In the d e r m i s s u r r o u n d i n g the scars, n o significant difference was f o u n d in vessel score b e t w e e n the scar types. Statistical c o m p a r i s o n with d e r m i s s u r r o u n d i n g p e r i o c u l a r scars was n o t possible b e c a u s e of the small a m o u n t of tissue available in s o m e cases. C o m p a r i n g r e c e n t surgical scars (less t h a n 35 days duration, n = 13) a n d older scars (35 or m o r e days
Actual VesselScoresfor al! Scar Types
Vessel Score*
Surgical scars
Periocular scars
Vessel Score*
Lesion
Dermis
Scar Duration (days)
0.96 2.55 2.23 2,55 1,59 0,96 1,75 0.48 1.11 1.11 3.98 2.O7 3.18 1.59 1.75 1.59 2.23 2.07 1.11 2.23 1.59 1.27 1.75 1.75 1.43 1.59 1.11 0.64 0.64 2.23 2.23 1.27 1.91 0.80 1.75 2.39
1.27 1.75 1.27 0.48 1.91 1.43 2.55 1.27 0132 0.64 8.76 1.75 0.96 0,80 0.48 1,59 2.87 1.59 0.32 0.96 2.07 1.59 2.39 1.75 1.43 ND ND ND ND ND ND 1.59 ND ND 2.39 ND
8 13 21 23 26 26 28 29 30 32 32 32 33 35 35 36 36 37 37 39 46 49 49 5O 83 49 ND ND ND ND ND ND ND ND ND ND
Scar Duration Lesion
Dermis
(too)
Hypertrophic scars
1.59 2.71 3.50 0.96 1.11 1.27 2.87 1.43 1.11 2.71
0.96 1.91 1.11 1.75 3.03 2.07 1.il 3.03 1.43 1.27
12 12 12 22 ND ND ND ND ND ND
Keloid scars
0.16 2.55 0.64 O.8O 0.80 0.80 1.27 !. 11 0.48 0.96 0.64 1.27
1.27 3.34 2.23 2.07 3.03 0.64 4.94 2.39 3.34 1.27 3.66 1.43
18 22 26 30 60 96 ND ND ND ND ND ND
Abbreviations: ND, no data available. *Vessel score = number of Chalkley point "hits" with endothelial ceils or vessel lumina per square millimeter.
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ANGIOGENESIS IN SCARS (Beer et al)
FIGURE 1. Hypertrophic scar showing a dense proliferation of fine vessels. (Immunohistochemical stain for factor VIII-related antigen; original magnification x 160.)
d u r a t i o n , n = 12), n o s i g n i f i c a n t d i f f e r e n c e in vessel scores was s e e n f o r e i t h e r l e s i o n a l o r d e r m a l tissue. However, r e c e n t scars d i d s h o w n e a r l y twice as m u c h variability in vessel s c o r e w h e n s t a n d a r d d e v i a t i o n s w e r e c o m p a r e d . T h e r e were insufficient d a t a to allow statistically valid c o m p a r i s o n o f vessel s c o r e a n d scar d u r a t i o n f o r t h e o t h e r scar types. W i t h i n s o m e o f t h e cases o f k e l o i d s c a r r i n g , t h e r e was a subjective r e d u c t i o n in vessel l u m e n a l d i a m e t e r . This a p p e a r e d to b e r e l a t e d to p r o m i n e n t e n d o t h e l i a l cells p r o j e c t i n g i n t o t h e l u m e n (Fig 3). T h e f i n d i n g was o f a focal n a t u r e , a n d n o f i b r i n d e p o s i t i o n o r t h r o m b u s were i d e n t i f i e d . I n c o n t r a s t , in the dermis immediately surrounding some of the nodules o f k e l o i d a l tissue, vessels a p p e a r e d d i l a t e d with a less c o n s p i c u o u s e n d o t h e l i a l lining. I n s o m e o f t h e h y p e r t r o p h i c scar cases, a s i m i l a r p a t t e r n o f fine vessels with p a r t l y o b s t r u c t e d l u m i n a was also n o t e d , a n d s u r r o u n d i n g v a s c u l a r d i l a t i o n was also s e e n in s o m e cases. S u r g i c a l scars g e n e r a l l y s h o w e d m o r e widely p a t e n t vessels t h a n k e l o i d o r h y p e r t r o p h i c scars, b u t in s o m e e x a m p l e s vessels with p r o m i n e n t e n d o t h e l i a l cells a n d o n l y a tiny l u m e n were i d e n t i f i e d . T h e surgical scars l a c k e d t h e p e r i p h e r a l v a s c u l a r d i l a t i o n s e e n in k e l o i d a n d h y p e r t r o p h i c scars. Mast cell n u m b e r s in a n d a r o u n d e a c h o f t h e s e scars h a d b e e n m e a s u r e d in a TABLE 2.
p r e v i o u s study. :° T h e r e was n o r e l a t i o n s h i p b e t w e e n t h e n u m b e r s o f m a s t cells a n d vessel s c o r e f o r a n y scar type. DISCUSSION This s t u d y p r o v i d e s q u a n t i t a t i v e e v i d e n c e t h a t kel o i d scars have a r e d u c e d vascular d e n s i t y c o m p a r e d with h y p e r t r o p h i c scars a n d surgical scars at p e r i o c u l a r a n d systemic sites. I n a d d i t i o n to a r e d u c e d vascular density, s o m e k e l o i d s s h o w e d subjective p a r t i a l occlus i o n o f vascular l u m i n a by p r o m i n e n t e n d o t h e l i a l cells. T h i s was s e e n in s o m e cases o f m a n y m o n t h s ' d u r a t i o n , suggesting that they do not merely represent new c a p i l l a r i e s j u s t b e g i n n i n g to f o r m h o l l o w l u m i n a . Simi-
3.5
3.0
~. 0
2.5
g 2.0
g
1.5
M e a n Vessel Score of the Four Scar Types
Mean Vessel Score* (Standard Deviation)
Keloid scars Surgical scars Hypertrophic scars Periocnlar scars
1.0
Lesion
Dermis
Ratio Mean Vessel Score Lesion: Dermis
0.96 (0.6) 1.80 (0.75) 1.93 (0.92) 1.51 (0.65)
2.47 (1.24) 1.69 (1.62) 1.77 (0.76) 1.99 (0.57)
0.39 1.07 1.09 0.76
.5 ¸
0.0. 11 Periocular
1~2 Hypertrophic
12 Keloid
25 Surgical
Scar type
*Vesselscore = number of Chalkley point "hits" with endothelial cells or vessel lumina per square millimeter.
FIGURE 2. Vessel scores of the four scar types with 95% confidence intervals.
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FIGURE3, Keloid scar showing a low vascular complement. Note the presence of a number of fine vessels with largely o c c l u d e d lumina (arrows). (Immunohistochemical stain for factor Viii-related antigen; original magnification x 160.)
larly narrowed vessels were also n o t e d in some hypertrophic and surgical scars. O t h e r groups have stated that hypertrophic or keloid scars have an increased vascular c o m p o n e n t compared with healing scars, 5-7 but these suggestions were based on qualitative studies with small numbers of cases. T h e concept of keloid hypervascularity has b e c o m e perpetuated in the literature 3,8 with suggestions that angiogenic factors from macrophages may enhance the growth of keloids. 11 Mast cells may also have an angiogenic action, le but our previous findings have questioned the role of mast cells in stimulating keloid development. 1° Microvascular occlusion in hypertrophic scars and keloids has been well documented, 5,7,8,1s although this change is not confined to pathological scars. One of the key studies in this area f o u n d that occluded vessels were present in very similar numbers in keloids, mature scars, and normal skin. 5 Recently, a correlation between myocardial fibrosis and capillary Reduced angiogenesis
\
Wound tension / infection
FIGURE4. Hypoxia is of crucial importance in the development of keloid scars.
luminal narrowing has b e e n d o c u m e n t e d in hypertrophic hearts. 14 Vascular dilation at the peripheral margins of the lesions was noted only for hypertrophic scars and keloids. Vessel numbers in histologically normal dermis surrounding the scars were similar for all scar types. Keloid and hypertrophic scars are known to be hypoxic, 7 and we postulate that in keloids hypoxia is mediated through their reduced vascularity c o m b i n e d with microvascular occlusion. O t h e r factors are clearly also important, but hypoxia is likely to play a crucial role (Fig 4). Keloids occurring at sites of skin tension and in wounds that have been infected or u n d e r tension are well documented, and these circumstances may cause tissue hypoxia. Although hypoxia appears important in the development and maintenance of keloids, severe or "critical hypoxia" may cause their involution. Prolonged external pressure applied to keloids is known to reduce scar severity, 15A6 and this could be mediated
Abnormal endothelial cells obstructing vessel lumen
E HYPOXIA
~
KELOID FORMATION
I
Sites of skin tension
Irradiation External pressure
Excess collagen deposition by fibroblasts Critical hypoxia
1 1
Fibroblast death
Keloid involution
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ANGIOGENESIS IN SCARS (Beer et al)
through vascular compression resulting in fibroblast death from ischemia. Irradiation of keloids may also reduce their severity, 17 possibly through ischaemia produced by microvascular damage. Excess collagen deposition by fibroblasts in keloids 6,7'n'18'19 inhibits oxygen diffusion, promoting tissue hypoxia and further collagen production. Transforming growth factor-beta (TGF-[3) promotes increased collagen production by fibroblasts and is increased in keloids and hypertrophic scars. 2°,21 The crucial role of TGF-[3 has b e e n demonstrated by neutralizing its effects with specific antibodies, which leads to decreased collagen production and reduced scarring. 18,21-23 Scarless healing in fetal tissue is characterized by an absence of TGF-[~ at the wound site. 24 The role of TGF-[3 in angiogenesis is unclear 25 and warrants further investigation. The reduced vascularity of keloids may also promote collagen deposition through a lack of the inhibitory action of factor XIIIa-positive dermal dendrocytes. Keloids are known to have a low c o m p l e m e n t of these cells, 2e-28 which inhibit collagen synthesis 28-~° and are closely associated with vessels. 3~,~2Factor XIIIa has also been localized to endothelial cells by electron microscopy? 3 Mast cells may promote collagen deposition 11,18 and angiogenesis 12,~4,3~ mediated by heparin, but we could not confirm a relation between mast cells and angiogenesis in these types of scar. In a previous study, 1° we have f o u n d no difference in mast cell numbers between keloids, hypertrophic scars, and surgical scars in nonperiocular sites. Periocular tissue scars were, however, f o u n d to have increased numbers of mast cells c o m p a r e d with all other scar types.
CONCLUSION Keloids show a quantitative reduction in vessels compared with hypertrophic and surgical scars at periocular and other sites. This helps to explain their hypoxic nature, especially because there may be some occlusion of the scant vessels present. Vascular quantification may help in making the important distinction between hypertrophic and keloid scars, ~ and this m e t h o d goes some way to realizing the goal of a laboratory test to separate the two as envisaged by Norris. 37 Keloid scar formation is associated with tissue hypoxia, which is partly mediated by reduced vascularization.
Acknowledgment. T h e a u t h o r s t h a n k R. M a n n e r s for s u p p l y i n g m a t e r i a l f r o m t h e p e r i o c u l a r scars a n d to L. West for t e c h n i c a l assistance. REFERENCES I. Chrisman BB: Blepharoplasty and browlift with surgical variations in non-white patients.J Dermatol Surg Oncol 12:58-66, 1986 2. Offret H, Quillard J: Nodule cheloidien du bord libre de la paupiere.J Fr Ophthalmo116:552-553, 1993 3. Rockwell WB, Cohen IK, Ehrlich HP: Keloids and hypertrophic scars: A comprehensive review. Plast Reconstr Surg 84:82%837, 1989
4. McGrouther DA: Hypertrophic or keloid scars? Eye 8:200-203, 1994 5. Kischer CW, Thies AC, Chvapil M: Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids. HuM PATHOL 13:819-824, 1982 6. Thomas DW, Hopkinson I, Harding KG, et ah The pathogenesis of hypertrophic/keloid scarring. Int J Oral MaxiIlofac Surg 23:232-236, 1994 7. Kischer CW: The microvessels in hypertrophic scars, keloids and related lesions: A review. J Submicrosc Cytol Pathol 24:281-296, 1992 8. Ehrlich HE Desmouliere A, Diegelmann RF, et ah Morphological and immunochemical differences between keloid and hypertrophic scar. AmJ Patho1145:105-113, 1994 9. McKee PH (ed): Pathology of the Skin with Clinical Correlations (ed 2). London, England, Mosby-Wolfe, 1996, pp 16.8-16.9 10. Beer TW, Baldwin H, West L, et al: Mast cells at pathological and surgical scar sites. BrJ Ophthalmo182:691-694, 1998 11. Smith CJ, Smith JC, Finn MC: The possible role of mast cells (allergy) in the production of keloid and hypertrophic scarring. J Burn Care Rehabil 8:126-131, 1987 12. Meininger CJ, Zetter BR: Mast Cells andAngiogenesis. Semin Cancer Biol 3:73-79, 1992 13. Lametschwandtner A, Staindl O: The vascular architecture of the keloid: A scanning electron microscope study on vascular corrosion casts. HNO 38:202-207, 1990 14. Takemura G, Takatsu Y, Fujiwara H: Luminal narrowing of capillaries in human hypertrophic hearts: An ultrastructural morphometrical study using endomyocardial biopsy specimens. Heart 79:7885, 1998 15. Zuber TJ, DeWitt DE: Earlobe keloids. Am Faro Physician 49:1835-1841, 1994 16. Murray JC: Scars and keloids. Dermatol Clin 11:69%708, 1993 17. NorrisJE: Superficial x-ray therapy in keloid management: A retrospective study of 24 cases and literature review. Plast Reconstr Surg 95:1051-1055, 1995 18. Suzawa H, Kikuchi S, Arai N, et al: The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts.JpnJ Pharmaco160:91-96, 1992 19. Riaz Y, Cook HT, Wangoo A, et al: Type 1 procollagen as a marker of severity of scarring after sternotomy: Effects of topical corticosteroids. J Clin Pathol 47:892-899, 1994 20. Zhang K, Garner W, Cohen L, et al: Increased types I and III collagen and transforming growth factor-beta 1 mRNA and protein in hypertrophic burn scar.J Invest Dermato1104:750-754, 1995 21. Border WA, Nobe NA: Transforming growth factor [3 in tissue fibrosis. N EnglJ Med 331:1286-1292, 1994 22. Shah M, Foreman DM, Ferguson MWJ: Neutralisation of TGF-beta 1 and TGF-beta2 or exogenous addition of TGF-beta3 to cutaneous rat wounds reduces scarring. J Cell Sci 108:985-1002, 1995 23. Ferguson MWJ: Skin wound healing: Transforming growth factor beta antagonists decrease scarring and improve quality. J Interferon Res 14:303-304, 1994 24. Nath RK, LaRegina M, Markham H, et ah The expression of transforming growth factor type beta in fetal and adult rabbit skin wounds.J Pediatr Surgy 29:416-421, 1994 25. Levine JH, Moses HL, Gold LI, et al: Spatial and temporal patterns ofimmunoreactive transforming growth factor 131,[32,and [33 during excisional wound repair. AmJ Patho1143:368-380, 1993 26. Altman DA, Nickoloff BJ, Fivenson DP: Differential expression of factor XIIIa and CD34 in cutaneous mesenchymal tumours. J Cutan Pathol 20:154-158, 1993 27. Cerio R, SpaullJ, Jones EW: Histiocytoma cuffs: A tumour of dermal dendrocytes (dermal dendrocytoma). BrJ Dermatol 120:197206, 1989 28. Pierard GE, Arrese-EstradaJ, Pierard-Franchimont C, et al: Is there a link between dendrocytes, fibrosis and sclerosis? Dermatologica 181:264-265, 1990 29. Pierard-Franchimont C, Arrese JE, Nikkels AF, et al: Factor XIIIa-positive dendrocytes and proliferative activity of cutaneous cancers. Virchows Arch A Pathol Anat Histopathol 429:43-48, 1996
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30. Hoyo E, Kanitakis J, Schmitt D: The dermal dendrocyte. Pathol Bio141:613-618, 1993 31. Suvarna SK, Cotton DWK: Dermal dendrocytes and other factor XIIIA-positive cells.J Pathol 171:251-252, 1993 32. Penneys NS: Factor XIII expression in the skin: Observations and a hypothesis.J Am Acad Dermatol 22:484488, 1990 33. Schaumburg-Lever G, Gehring B, Kaiserling E: Ultrastructural localisation of factor XIIIa. J Cutan Pathol 21:129-134, 1994
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34. Trabucchi E, Radaelli E, Marazzi M, et al: The role of l~last cells in wound healing. IntJ Tissue Reac X:367-372, 1988 35. Arbiser JL: Angiogenesis and the skin: A primer. J Am Acad Dermato134:486-497, 1996 36. Brody GS: Keloids and hypertrophic scars. Plast Reconstr Surg 86:804, 1990 37. NorrisJE: Hypertrophic scar and keloid [letter]. Plast Reconstr Surg 88:372-373, 1991