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Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids
PERIVASCULAR MYOFIBROBLASTS AND MICROVASCULAR O C C L U S I O N IN H Y P E R T R O P H I C SCARS A N D KELOIDS C. Ward Kische~, PhD,* A. Cole Thies,'~ and Milos Chvapil, MD, PhD$ Microvessels in normal skin, granulation tissue, hypertrophic scar, keloid, and mature s c a r f r o m h u m a n subjects were studied by transmission e l e c t r o n m i c r o s c o p y . Comparative observations s u g g e s t e d that most microvessels in hypertrophic scar a n d keloid are o c c l u d e d o r partially occluded, apparently o w i n g to an e x c e s s o f endothelial cells. Endothelial cell contraction was also supported by the observations, and perivascular satellite cells (pericytes), some of which were identified as myofibroblasts, w e r e observed in hypertrophic scars a n d keloids. A m o n g findings f r o m statistical analyses were that 1) the pateney of microvessels in hypertrophic scar and granulation tissue is similar, as is that o f microvessels in keloid and mature scar, but the patency of all these microvessels is significantly less than that o f microvessels in normal skin, and 2) endothelial cell density is greater in nonpatent vessels than in patent vessels. The observed e x t e n t o f micrnvascular occlusion supports a previously published theory that hypoxia is involved in the generation of hypertrophic scar. H u m Pathol 13:819-824, 1982.
ha one o f o u r early studies o f h y p e r t r o p h i c scar we observed microvessels (capillaries) with occluded lumens. ~ This p h e n o m e n o n was observed again in later studies. 2-4 T h e occlusion a p p e a r e d to be the result o f an excessive n u m b e r o f endothelial cells that bulged into the lumen. W h e n we suggested in an earlier publication that c h r o n i c hypoxia might be involved in the d e v e l o p m e n t o f h y p e r t r o p h i c scar, 2 tt~e possibility o f extensive microvascular occlusion assunted greater significance, since such a condition could account for the suspected hypoxia. Recently, Sloan et al. ~ verified hypoxia in h y p e r t r o p h i c scar. For the past two ),ears we have d e l i b e r a t e l y screened every sample available to us o f h y p e r t r o p h i c scar, keloid, m a t u r e scar, and granulation tissue for ultrastructural characterization o f the microvessels. In addition, we have secured and screened samples o f normal skin. This p a p e r reports an assessment o f the microvessels for each g r o u p as d e t e r m i n e d by statistical analysis. MATERIALS AND METHODS
H u m a n tissues were obtained from surgical excision procedures. T h e tissue samples included 11 o f Received from the Departments o f Anatomy,* C o m p u t e r Systems and Biostatistics,t and Surgery,:]: University o f Arizona College o f Medicine, Tucson, Arizona. Accepted for pnblication j a n u a r y 8, 1982. Supported in part by National Institutes of Health research grant 1-ROIGM25159. Address correspondence and reprint requests to Dr. Kischer, D e p a r t m e n t o f A n a t o m y , University o f A r i z o n a College o f Medicine, Health Sciences Center, Tucson, A Z 85724.
normal skins, 13 o f h y p e r t r o p h i c scar, nine o f keloid, five o f m a t u r e scar, and nine o f granulation tissue. T h e s e tissues were dissected appropriately, fixed immediately in Karnovsky's solution, and stored from two hours to several days. Cores 1 m m in d i a m e t e r were obtained by means o f a modilied Jahmslfidi liver p u n c h and washed in isotonic cacodylate b u f f e r at p H 7.4. Tile tissues were then postfixed in isotonic osm i u m tetroxide. A f t e r ethanolic d e h y d r a t i o n , the tissues were e m b e d d e d in Epon 812. T h i c k sections were cut at 1 /.tm and stained with tohfidine blue. T h i n sections were stained with uranyl acetate and e x a m i n e d in a Philips 300 transmission electron microscope. T h e s e tissues were e x a m i n e d primarily for evaluation o f the microvasculature. T h e diameters o f the microvessels r a n g e d f r o m 3.3 to 14.6 #m, which is the normal range. Rarely was any larger vessel e n c o u n t e r e d i n any tissue g r o u p , except n o r m a l skin. T h e u h r a s t r u c t u r a l clmracterization o f the microvessels included d e t e r m i n a t i o n o f I) hmtinal patency (or occlusion), 2) the n u m b e r s o f endothelial cell and nuclear profiles, 3) contraction o f endothelial cells, det e r m i n e d largely by endothelial nuclear crenation, and 4) types and conditions o f perivasctdar satellite cells (pericytes). T h e following n u m b e r s o f microvessels were studied in each g r o u p : 25 in n o r m a l skin, 76 in granulation tissue, 306 in h y p e r t r o p h i c scar, 106 in keloid, a n d 51 in m a t u r e scar. Only microvessels cut unambiguously in cross-sections (usually r o u n d in profile) were used in the data collection. O n l y a r a r e view o f c r o s s - s e c t i o n e d m i c r o v e s s e l suggested collapse o f the wall. No confusion existed in distinguishing vessel collapse from noncollapse. Full occlusion was based on a lumen less than 1 /.tin in diameter. Partial occlusion was assessed for lumens up to 3 / ~ m across; b e y o n d that the microvessel was considered to be patent. T h e p a t e n c y d a t a w e r e e x a m i n e d u s i n g chisquare statistics d e t e r m i n e d f r o m contingency tables. In addition, 95 per cent confidence intervals were d e t e r m i n e d for each tissue group. Endothelial cell density was assessed across tissue g r o u p s and was c o m p a r e d b e t w e e n p a t e n t a n d n o n p a t e n t vessels using a two-way analysis o f variance. F u r t h e r characterizations were based o n comparative quantities in the endothelial cells o f I) microfilarnents, 2) Weibel-Palade bodies, 3) microvesicular bodies, 4) mitochondria, 5) Golgi m e m b r a n e s , and 6) r o u g h endoplasmic reticulum. Gradings on
0046-8177/82/090010819 $01.20 9 W. B. Saunders Co.
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HUMAN PATHOLOGY~VOLUME 13, NUMBER 9 September1982 !
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Figure 1. Capillaryfrom dermis of normal skin. Two endothelial cells are evident and celljunctions are simple. Endothelial cytoplasm contains few organelles but many micropinocytotic vesicles on both luminal and abluminal sides. Sections of a likely single pericyte are present with essentially the same void of organelles as the endothelium. (• 19,500.)
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a scale o f 0 to 5 were assigned arbitrarily f r o m study o f electron micrographs. RESULTS
Electron Microscopy Figure 1 illustrates the typical fine s t r u c t u r e o f a capillary f r o m n o r m a l skin. In this section, profiles o f a p p a r e n t l y two endothelial cells can be seen. T h e junctions o f the endothelial cells are relatively simple. Endothelial cytoplasm contains a few microfilaments, a scattering o f free ribosomes, an occasional p r o f i l e o f r o u g h e n d o p l a s m i c r e t i c u l u m , b u t an a b u n d a n c e o f micropinocytotic vesicles on both the luminal a n d a b l u m i n a l sides. U n d e r l y i n g the end o t h e l i u m is a s i m p l e basal l a m i n a c o m p r i s i n g g r a n u l a r and short filamentous structures approximately 30 n m thick. Outside this layer but encased in their own basal lamina are the pericytes. In any given cross-section, a profile o f usually just one pericyte cell will be seen. T h e cells closely resemble endothelial cells, although we have n e v e r observed Weibel-Palade bodies in them. Occluded capillaries were sometimes observed in n o r m a l skin. Usually these o c c u r r e d in so-called normal samples taken adjacent to h y p e r t r o p h i c scar lesions. O c c l u d e d capillaries p r e d o m i n a t e d in h y p e r trophic scar and keloid. Only occasionally were patent vessels o b s e r v e d in this g r o u p . F i g u r e 2 shows a cross-section o f a capillary representative o f those in h y p e r t r o p h i c scar. It is 8 # m in diameter. O b s e r v e d in this section are profiles o f at least 12 endothelial cells,
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including t h r e e e n d o t h e l i a l nuclei. T i l e l u m e n is occluded. Note also the high position o f the tight junctions between the endothelial cells. T h e r e are a few organelles within the endothelial cytoplasm, but, although not seen at the magnification o f figure 2, the cytoplasm is rich in microfilaments. Endothelial nuclei a r e essentially r o u n d . S u r r o n n d i n g t h e endothelium is a single layer o f basal lamina. In most cases, however, the basal lamina is t h i c k e n e d and multi-layered. Also seen in figure 2, a satellite cell nearly circumscribes tile entire p e r i p h e r y o f the e n d o t h e l i u m . T h e i n n e r rim o f this satellite cell displays a band o f microfilaments that is aligned parallel to the circumference o f the vessel and periodically shows cytoplasmic densities (arrows). Such a band is n e v e r seen at the p e r i p h e r y o f a cell. No similar satellite cells have been observed in n o r m a l dermis or m a t u r e scar. We interpret these pericytes as myofibroblasts. In o t h e r occluded microvessels, endothelial nuclei a p p e a r e d highly lobated o r crenated. In figure 3, five profiles o f endothelial cells can be seen. Outside this layer is a c o m p l e t e basal lamina. T h e satellite cell again shows the i n n e r band o f microfilaments and cytoplastnic densities. Outside the satellite cell is a multilayered basal lamina. C o m p l e x unions between endothelial cells were o f t e n observed (fig. 4). Despite this, tight j u n c t i o n s or gap junctions were virtually always seen and in most instances were positioned high on the cell boundaries. No wide-open breaks o r gaps have yet been observed. T h e endothelial cells in h y p e r t r o p h i c scars usually contain notable axnounts o f microfilaments, increased n u m b e r s o f mitochondria, Golgi m e m b r a n e s ,
MICROVESSELS AND SCAR--Kxsct~ER F.T AL.
Figure 2. Cross-section of microvessel from hypertrophic scar. Profiles of at least 12 endothelial cells are present. A satellite cell is present with an inner rim of microfilaments and cytoplasmic densities (arrows). Note also the high position of interendothelial tight junctions. (• 10,200.)
a n d profiles of r o u g h e n d o p l a s m i c reticulum. T h e numbers of Weibel-Palade bodies and multi-vesicular b o d i e s d i d n o t a p p e a r to d i f f e r f r o m t h o s e i n all o t h e r g r o u p s , i n c l u d i n g n o r m a l d e r m i s . A l t h o u g h vesicles
w e r e n o t c o u n t e d , we o b s e r v e d t h a t t h e r e a r e u s u a l l y f e w e r m i c r o p i n o c y t o t i c vesicles in e n d o t h e l i a l a n d satellite cells o f h y p e r t r o p h i c scar a n d k e l o i d c o m p a r e d with n o r m a l d e r m i s .
Figure 3. Partially occluded microvessel from hypertrophic scar. Note crenated endothelial nucleus. Five endothelial cells are present. The satellite cell shows an inner rim of microfilaments and cytoplasmic densities. The basal lamina is complex and thickened. (•
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HUMAN PATHOLOGY---VOLUME 13, NUMBER 9 September 1982
Figure 4. Complex endothelial junctions in occluded vessel from hypertrophic scar (arrows). ( x 18,000.)
Statistical Analysis T h e d i a m e t e r o f the microvessels in the tissues e x a m i n e d varied f r o m 3 to 15 btm, but the majority were between 7 a n d 9/~ms. T a b l e 1 shows the m e a n s a n d ranges o f d i a m e t e r s for all vessels studied f r o m the several g r o u p s . All were o f equivalent size. T h e results o f chi-square statistical analyses o f the patency frequencies for all g r o u p s are s h o w n in table 2. Patency rates w e r e a p p r o x i m a t e l y 60 p e r cent f o r n o r m a l skin, 7 p e r cent for g r a n u l a t i o n tissue a n d h y p e r t r o p h i c scars, 17 p e r cent for keloid, a n d 20 p e r cent for m a t u r e scar. Chi square analysis o f the data shown in table 2 indicated statistical significance (P < 0.001). E x a m i n i n g a 95 p e r cent c o n f i d e n c e interval for p a t e n c y across tissue g r o u p s , we c o n f i r m e d t h a t g r a n u l a t i o n tissue is similar to l l y p e r t r o p h i c scar a n d that keloid is similar to m a t u r e scar. Each g r o u p indicated a patency rate significantly lower t h a n that o f n o r m a l skin. T a b l e 3 shows the density o f endothelial cells in all microvessel profiles o b s e r v e d . A two-way analysis o f variance using the n u m b e r o f endothelial cell p r o files p e r s q u a r e m i c r o m e t e r yielded the following results: 1) patent vessels showed significantly less endothelial density t h a n n o n p a t e n t vessels (P = 0.01); 2) controlling for patency, we f o u n d no significant differences a m o n g the tissue g r o u p s (P = 0.13); a n d 3) t h e r e was no significant interaction between p a t e n c y a n d tissue g r o u p (P = 0.89).
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Analysis o f variance data for the n u m b e r s o f nuclear profiles i n d i c a t e d no significant d i f f e r e n c e s a m o n g the tissue g r o u p s studied. DISCUSSION
T i l e vast m a j o r i t y o f ttle m i c r o v e s s e l s in the h y p e r t r o p h i c scars a n d keloids had occluded lumens. T h i s f i n d i n g c o n t r a s t e d s h a r p l y with t h a t o f microvessels o b s e r v e d in n o r m a l skin. T h e fact that occluded microvessels were s o m e t i m e s o b s e r v e d in some o f o u r n o r m a l samples m a y reflect o u r inability to obtain truly n o r m a l skin samples. Most o f o u r specimens were f r o m areas ixnmediately adjacent to a lesion or were o b t a i n e d f r o m lipectomies. T h e microvessels o f the h y p e r t r o p h i c scars a n d o f m a n y keloids very often a p p e a r e d to have several satellite cells a b o u t t h e m . T h i s c o n t r a s t e d with what is usually o b s e r v e d for a capillary, w h e r e o n e perivas-
T A B L E 1.
DIAMETERS
OF MICROVESSELS
Mean __ SD
Normal skin Granulation
(n = 25)
tissue (n = 76) Hypertrophic scar (n = 306) Keloid (n = 106) Mature scar (n = 51)
(#m)
Minimum Maximum
8.2 -+ 2.7
3.3
13.9
9.2 - 2.3
4.5
13.8
8.3 - 2.3 7.8 -+ 2.4 7.7 - 2.2
3.3 3.7 3.8
14.6 14.5 13.3
MICROVESSELS AND SCAR--KlsCHER ET AL. TABLE 2.
Normal skin Granulation tissue Hypertrophic scar Keloid Mature scar
(n (n (n (n (n
= 25) = 75]') = 306) = 105i) = 51)
PATENCY OF MICROVESSELS* 95% Confidence Interval for Patency (%)z~
Patency
Partial
FuI1
(%)
Occlusion (%)
Occlusion (%)
Low
High
60 6.6 6.9 17.1 19.6
36 30.7 19.3 20.0 33.3
4 62.7 73.8 62.9 47.1
39 2 4 11 10
79 15 11 26 33
* X~ = 51.7, P < 0.001 (see text for discussion). i Data not determined for one case. :]:Documenta, Geigy. Scientific Tables, 6th Editiou, page 86, 1962. TABLE 3. No. of Vessels
Normal skin Granulation tissueqt Hypertroplfic scar Keloid Mature scar
Patent
Occludedt
15 5 21 18 10
10 70 285 87 40
ENDOTHELIAL CELL DENSITY (per/xm2) * Mean density (• Patent
0.09 0.07 0.10 0.10 0.13
_ • • • _
0.04 0.03 0.05 0.05 0.05
Minimal Density
Occluded
0.13 0.10 0.14 0.14 0.14
• • • • •
0.05 0.05 0.07 0.09 0.07
Maximal Density
Patent
Occluded
Patent
Occluded
0.05 0.04 0.03 0.03 0.08
0.07 0.03 0.03 0.03 0.05
0.15 0.11 0.19 0.24 0.26
0.23 0.29 0.53 0.52 0.33
* Results of two-way analysis of variance: effect of patency significant (P = 0.01); effect of tissue not significant (P = 0.13); no interaction between patency and tissue (P = 0.89). (See text for further discussion.) i" Includes partial and full occlusion. Data not available for one case.
cular satellite cell o r n o n e m a y be presentY. In m a n y cases the i n n e r m o s t satellite cell had every characteristic o f a myofibroblast. In any event, no previous r e p o r t k n o w n to us identifies a myofibroblast as a pericyte o f capillaries. T h e occlusion we r e p o r t may be d u e to any o f three possible mechanisms, o r combinations o f these: I) perivascular myofibroblast contraction, 2) e n d o thelial cell contraction, or 3) endothelial cell proliferation. Pericapillary myofibroblasts were o b s e r v e d in about half o f the h y p e r t r o p h i c scars a n d keloids we studied. It may be that they eventually migrate f r o m the perivascular position to the interstitial area, w h e r e they are m u c h m o r e n u m e r o u s . A f t e r a time they may be t r a n s f o r m e d into fibroblasts. Pericapillary satellite cells are suspected o f being the source o f new fibroblasts in d e r m a l w o u n d s 7-9 and, possibly, .liver cirrhosis? ~ I n d e e d , o u r first study o f g r a n u l a t i o n tissue s h o w e d a p l e t h o r a o f p e r i c y t e s a b o u t the n e w l y g r o w i n g capillaries? T h e s m o o t h c o n t o u r o f nuclear sections t h r o u g h the pericapillary myofibroblast is not c o n s i d e r e d equivocal to a p r o p o s e d active c o n t r a c tion, since only the i n n e r o r vascular side o f the cytoplasm c o n t a i n s the m i c r o f i l a m e n t s a n d densities. F u r t h e r m o r e , active contraction o f the myofibroblast, which would have a direct functional effect o n the microvessel, is suggested because the microfilaments have not been observed elsewhere in the cell. Evidence o f endothelial cell contraction, which might contribute to luminal occlusion, has not been consistently observed. C r e n a t e d nuclei are the excep-
tion, not the rule. A l t h o u g h endothelial cell contraction, as previously r e p o r t e d , n-~3 would tend to shorten a vascular segment, a bulging o f tile nuclear region could p r o t r u d e into the lumen, thereby effecting an occlusion. However, we have f o u n d no consistent evidence o f s h o r t e n e d vascular segments by multiple light microscopy studies o f serial paraffin sections. By far the most plausible mechanism for occlusion can be s u p p o r t e d by the density o f profiles o f endothelial cells observed in cross-sections. T h e endothelial cell density data also strongly a r g u e against occlusion d u e to collapse. Besides, collapse is not really tenable in light o f r o u n d profiles o f microvessels. F u r t h e r m o r e , figure 2, in which a microvessel 8/~m in d i a m e t e r is occluded, showing profiles o f 12 e n d o thelial cells, also d e m o n s t r a t e s tight endothelial j u n c tions situated high between endothelial cells. This was a c o m m o n observation; it would not o c c u r if the endothelial cell density increased at a given focus o f the vessel owing to either contraction or endothelial cell collapse. It also is not reasonable to suspect that tim occluded microvessels in h y p e r t r o p h i c scar a n d keloid are simply the terminal closed ends o f g r o w i n g vessel buds. I n h y p e r t r o p h i c scars a n d keloids o f s o m e years' standing, this activity, expected in g r a n u l a t i o n tissue, would have long since ceased. Occlusion is essentially the same process in each g r o u p , as j u d g e d f r o m the interaction statistic. T h e density data also suggest that some n o r m a l cases were probably not normal. I n d e e d , some o f those cases were probably " n o r m a l " tissue immediately adjacent to the scar or keloid lesion.
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September1982
It is likely that e n d o t h e l i a l cell p r o l i f e r a t i o n is t h e m a j o r f a c t o r c o n t r i b u t i n g to occlusion. H o w e v e r , it is also likely t h a t p e r i v a s c u l a r m y o f i b r o b l a s t a n d e n d o thelial cell c o n t r a c t i o n s also c o n t r i b u t e , albeit minimally. T h e u l t r a s t r u c t u r e o f m o s t e n d o t h e l i a l cells in h y p e r t r o p h i c scar a n d keloid is in c o n c e r t with an active biologic state. W h e t h e r this indicates p r o l i f e r a tion c a n n o t be definitely stated here. H o w e v e r , p r o liferation c o u l d be s u p p o r t e d by i n c r e a s e d n u m b e r s o f o r g a n e l l e s a n d m a n y intercellular j u n c t i o n s t h a t f o r m c o m p l e x u n i o n s o r m a z e s o f intricate partially i n t e r d i g i t a t e d cell processes. It is o f special significance that in o u r p r e v i o u s s t u d y o f a first series o f g r a n u l a t i o n tissues f r o m d e e p h u m a n w o u n d s a we f o u n d o c c l u d e d microvessels o n l y in o l d e r g r a n u l a t i o n s , t h o s e in w h i c h t h e c o l l a g e n patterns of hypertrophic nodules had already been well established. A d d i t i o n a l l y , fibrin p o l y m e r was persistent in e v e r y case e x a m i n e d . It a p p e a r s likely t h a t m i c r o v a s c u l a r o c c l u s i o n in h y p e r t r o p h i c scars a n d keloids is d u e principally to i n c r e a s e d n u m b e r s o f e n d o t h e l i a l cells. It m a y also be t h a t a p r o l i f e r a t i o n is established in t h e g r a n u l a t i o n phase. T h e factors p r o m o t i n g a s u s p e c t e d p r o l i f e r a tion are n o t k n o w n ; h o w e v e r , b e c a u s e we h a v e obs e r v e d pervasive a n d persistent fibrin in o u r series o f g r a n u l a t i o n tissues, we s u g g e s t that this is a possible s o u r c e o f stimulation. Fibrin has b e e n i d e n t i f i e d as stimulating a marked uptake ofSH-labeled thymidine in c u l t u r e s o f fibroblasts, t4"t5 O n c e again, we a r e c o m p e l l e d to r e a f f i r m o u r h y p t h e s i s t h a t h y p o x i a is an i n t e g r a l f a c t o r in t h e g e n e r a t i o n o f h y p e r t r o p h i c scar a n d keloid. W e h a v e provided strong morphologic evidence of the origin o f t h e h y p o x i a . W h e t h e r the h y p o x i a m a y stimulate p r o l i f e r a t i o n o f e n d o t h e l i a l cells, pericytes, a n d fibroblasts o r the synthesis o f collagen, o r all, r e m a i n s to be d e t e r m i n e d . H u n t et al. ~6 a r g u e c o n v i n c i n g l y f o r
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h y p o x i a c o n t r o l l i n g collagen synthesis in the w o u n d s o f rabbits a n d dogs.
REFERENCES I. Kischer CW, Linares H, Dobrkovsky M, et al: Electron microscop)' of the hypertrophic scar. Twenty-ninth Annual Proceedings of the Society of America, 1971, p 302. 2. Kischer CW, Shetlar MR, Shet[ar CL: Alterations of hypertrophic scars induced by mechanical pressure. Arch Dermatol I11:60, 197.6 3. Kischer CW: Fine structure of granulation tissues from deep injury. J Invest Dermatol 72:147, 1979 4. Kischer CW, Shetlar MR: Microvasculature in hypertrophic scars and the effects of pressure. J Trauma 19:757, 1979 5. Sloan DF, Brown RD, Wells CH: Tissue gases in human hypertrophic burn scars. Plast Reconstr Surg 61:431, 1978 6. CliffWJ: Blood Vessels. London, Cambridge University Press, 1976, pp 68-72 7. Weber K, Braun-Falco O: Ultrastructure of blood vessels in human granulation tissue. Arch Dermatol Fortschr 248:29, 1973 8. Crocker DJ, Murad TM, Greer JC: Role of the pericyte in wound healing: an uhrastructural study. Exp Mol Pathol 13:51, 1970 9. Ross R, Everett NB, Tyler R: Wound healing and collagen formation. VI. The origin ofthe wound fibroblast studied in parabiosis. J Cell Biol 44:645, 1970 10. hie C, Kocher O, Gabbiani G: Contractility of myofibroblasts during experimental liver cirrhosis. J Submicrosc Cytol 12:209, 1980 11. Becker CG, Nachman RL: Contractile proteins of endothelial cells, platelets anti smooth muscle. Am J Pathol 71:1, 1973 12. Cliff, Wj: Blood Vessels. London, Cambridge University Press, 1976, pp 154-155 13. Majno G, Shea SM, Leventhal M: Endothelial contraction induced by histamine-type mediators.J Ceil Bio142:647, 1969 14. Nozawa RT: Altered morphology and increased cell adhesiveness of Chinese hamster ovary cells cultured on fibrin. J Cell Physiol 90:351, 1977. 15. Kittlick PD: Fibrin in fibroblast cultures: a metabolic study as a contribution to inflammation and tissue repair. Exp Pathol 17:312, 1979 16. Hunt TK, ConoIly WB, Aronson SB, et al: Anaerobic metabolism and wound healing: an hypothesis for the initiation and cessation of collagen synthesis in wounds. Am J Surg 135:328, 1979
ha one o f o u r early studies o f h y p e r t r o p h i c scar we observed microvessels (capillaries) with occluded lumens. ~ This p h e n o m e n o n was observed again in later studies. 2-4 T h e occlusion a p p e a r e d to be the result o f an excessive n u m b e r o f endothelial cells that bulged into the lumen. W h e n we suggested in an earlier publication that c h r o n i c hypoxia might be involved in the d e v e l o p m e n t o f h y p e r t r o p h i c scar, 2 tt~e possibility o f extensive microvascular occlusion assunted greater significance, since such a condition could account for the suspected hypoxia. Recently, Sloan et al. ~ verified hypoxia in h y p e r t r o p h i c scar. For the past two ),ears we have d e l i b e r a t e l y screened every sample available to us o f h y p e r t r o p h i c scar, keloid, m a t u r e scar, and granulation tissue for ultrastructural characterization o f the microvessels. In addition, we have secured and screened samples o f normal skin. This p a p e r reports an assessment o f the microvessels for each g r o u p as d e t e r m i n e d by statistical analysis. MATERIALS AND METHODS
H u m a n tissues were obtained from surgical excision procedures. T h e tissue samples included 11 o f Received from the Departments o f Anatomy,* C o m p u t e r Systems and Biostatistics,t and Surgery,:]: University o f Arizona College o f Medicine, Tucson, Arizona. Accepted for pnblication j a n u a r y 8, 1982. Supported in part by National Institutes of Health research grant 1-ROIGM25159. Address correspondence and reprint requests to Dr. Kischer, D e p a r t m e n t o f A n a t o m y , University o f A r i z o n a College o f Medicine, Health Sciences Center, Tucson, A Z 85724.
normal skins, 13 o f h y p e r t r o p h i c scar, nine o f keloid, five o f m a t u r e scar, and nine o f granulation tissue. T h e s e tissues were dissected appropriately, fixed immediately in Karnovsky's solution, and stored from two hours to several days. Cores 1 m m in d i a m e t e r were obtained by means o f a modilied Jahmslfidi liver p u n c h and washed in isotonic cacodylate b u f f e r at p H 7.4. Tile tissues were then postfixed in isotonic osm i u m tetroxide. A f t e r ethanolic d e h y d r a t i o n , the tissues were e m b e d d e d in Epon 812. T h i c k sections were cut at 1 /.tm and stained with tohfidine blue. T h i n sections were stained with uranyl acetate and e x a m i n e d in a Philips 300 transmission electron microscope. T h e s e tissues were e x a m i n e d primarily for evaluation o f the microvasculature. T h e diameters o f the microvessels r a n g e d f r o m 3.3 to 14.6 #m, which is the normal range. Rarely was any larger vessel e n c o u n t e r e d i n any tissue g r o u p , except n o r m a l skin. T h e u h r a s t r u c t u r a l clmracterization o f the microvessels included d e t e r m i n a t i o n o f I) hmtinal patency (or occlusion), 2) the n u m b e r s o f endothelial cell and nuclear profiles, 3) contraction o f endothelial cells, det e r m i n e d largely by endothelial nuclear crenation, and 4) types and conditions o f perivasctdar satellite cells (pericytes). T h e following n u m b e r s o f microvessels were studied in each g r o u p : 25 in n o r m a l skin, 76 in granulation tissue, 306 in h y p e r t r o p h i c scar, 106 in keloid, a n d 51 in m a t u r e scar. Only microvessels cut unambiguously in cross-sections (usually r o u n d in profile) were used in the data collection. O n l y a r a r e view o f c r o s s - s e c t i o n e d m i c r o v e s s e l suggested collapse o f the wall. No confusion existed in distinguishing vessel collapse from noncollapse. Full occlusion was based on a lumen less than 1 /.tin in diameter. Partial occlusion was assessed for lumens up to 3 / ~ m across; b e y o n d that the microvessel was considered to be patent. T h e p a t e n c y d a t a w e r e e x a m i n e d u s i n g chisquare statistics d e t e r m i n e d f r o m contingency tables. In addition, 95 per cent confidence intervals were d e t e r m i n e d for each tissue group. Endothelial cell density was assessed across tissue g r o u p s and was c o m p a r e d b e t w e e n p a t e n t a n d n o n p a t e n t vessels using a two-way analysis o f variance. F u r t h e r characterizations were based o n comparative quantities in the endothelial cells o f I) microfilarnents, 2) Weibel-Palade bodies, 3) microvesicular bodies, 4) mitochondria, 5) Golgi m e m b r a n e s , and 6) r o u g h endoplasmic reticulum. Gradings on
0046-8177/82/090010819 $01.20 9 W. B. Saunders Co.
8 19
HUMAN PATHOLOGY~VOLUME 13, NUMBER 9 September1982 !
9
9
,
...
Figure 1. Capillaryfrom dermis of normal skin. Two endothelial cells are evident and celljunctions are simple. Endothelial cytoplasm contains few organelles but many micropinocytotic vesicles on both luminal and abluminal sides. Sections of a likely single pericyte are present with essentially the same void of organelles as the endothelium. (• 19,500.)
9
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.
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,.
a scale o f 0 to 5 were assigned arbitrarily f r o m study o f electron micrographs. RESULTS
Electron Microscopy Figure 1 illustrates the typical fine s t r u c t u r e o f a capillary f r o m n o r m a l skin. In this section, profiles o f a p p a r e n t l y two endothelial cells can be seen. T h e junctions o f the endothelial cells are relatively simple. Endothelial cytoplasm contains a few microfilaments, a scattering o f free ribosomes, an occasional p r o f i l e o f r o u g h e n d o p l a s m i c r e t i c u l u m , b u t an a b u n d a n c e o f micropinocytotic vesicles on both the luminal a n d a b l u m i n a l sides. U n d e r l y i n g the end o t h e l i u m is a s i m p l e basal l a m i n a c o m p r i s i n g g r a n u l a r and short filamentous structures approximately 30 n m thick. Outside this layer but encased in their own basal lamina are the pericytes. In any given cross-section, a profile o f usually just one pericyte cell will be seen. T h e cells closely resemble endothelial cells, although we have n e v e r observed Weibel-Palade bodies in them. Occluded capillaries were sometimes observed in n o r m a l skin. Usually these o c c u r r e d in so-called normal samples taken adjacent to h y p e r t r o p h i c scar lesions. O c c l u d e d capillaries p r e d o m i n a t e d in h y p e r trophic scar and keloid. Only occasionally were patent vessels o b s e r v e d in this g r o u p . F i g u r e 2 shows a cross-section o f a capillary representative o f those in h y p e r t r o p h i c scar. It is 8 # m in diameter. O b s e r v e d in this section are profiles o f at least 12 endothelial cells,
820
including t h r e e e n d o t h e l i a l nuclei. T i l e l u m e n is occluded. Note also the high position o f the tight junctions between the endothelial cells. T h e r e are a few organelles within the endothelial cytoplasm, but, although not seen at the magnification o f figure 2, the cytoplasm is rich in microfilaments. Endothelial nuclei a r e essentially r o u n d . S u r r o n n d i n g t h e endothelium is a single layer o f basal lamina. In most cases, however, the basal lamina is t h i c k e n e d and multi-layered. Also seen in figure 2, a satellite cell nearly circumscribes tile entire p e r i p h e r y o f the e n d o t h e l i u m . T h e i n n e r rim o f this satellite cell displays a band o f microfilaments that is aligned parallel to the circumference o f the vessel and periodically shows cytoplasmic densities (arrows). Such a band is n e v e r seen at the p e r i p h e r y o f a cell. No similar satellite cells have been observed in n o r m a l dermis or m a t u r e scar. We interpret these pericytes as myofibroblasts. In o t h e r occluded microvessels, endothelial nuclei a p p e a r e d highly lobated o r crenated. In figure 3, five profiles o f endothelial cells can be seen. Outside this layer is a c o m p l e t e basal lamina. T h e satellite cell again shows the i n n e r band o f microfilaments and cytoplastnic densities. Outside the satellite cell is a multilayered basal lamina. C o m p l e x unions between endothelial cells were o f t e n observed (fig. 4). Despite this, tight j u n c t i o n s or gap junctions were virtually always seen and in most instances were positioned high on the cell boundaries. No wide-open breaks o r gaps have yet been observed. T h e endothelial cells in h y p e r t r o p h i c scars usually contain notable axnounts o f microfilaments, increased n u m b e r s o f mitochondria, Golgi m e m b r a n e s ,
MICROVESSELS AND SCAR--Kxsct~ER F.T AL.
Figure 2. Cross-section of microvessel from hypertrophic scar. Profiles of at least 12 endothelial cells are present. A satellite cell is present with an inner rim of microfilaments and cytoplasmic densities (arrows). Note also the high position of interendothelial tight junctions. (• 10,200.)
a n d profiles of r o u g h e n d o p l a s m i c reticulum. T h e numbers of Weibel-Palade bodies and multi-vesicular b o d i e s d i d n o t a p p e a r to d i f f e r f r o m t h o s e i n all o t h e r g r o u p s , i n c l u d i n g n o r m a l d e r m i s . A l t h o u g h vesicles
w e r e n o t c o u n t e d , we o b s e r v e d t h a t t h e r e a r e u s u a l l y f e w e r m i c r o p i n o c y t o t i c vesicles in e n d o t h e l i a l a n d satellite cells o f h y p e r t r o p h i c scar a n d k e l o i d c o m p a r e d with n o r m a l d e r m i s .
Figure 3. Partially occluded microvessel from hypertrophic scar. Note crenated endothelial nucleus. Five endothelial cells are present. The satellite cell shows an inner rim of microfilaments and cytoplasmic densities. The basal lamina is complex and thickened. (•
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HUMAN PATHOLOGY---VOLUME 13, NUMBER 9 September 1982
Figure 4. Complex endothelial junctions in occluded vessel from hypertrophic scar (arrows). ( x 18,000.)
Statistical Analysis T h e d i a m e t e r o f the microvessels in the tissues e x a m i n e d varied f r o m 3 to 15 btm, but the majority were between 7 a n d 9/~ms. T a b l e 1 shows the m e a n s a n d ranges o f d i a m e t e r s for all vessels studied f r o m the several g r o u p s . All were o f equivalent size. T h e results o f chi-square statistical analyses o f the patency frequencies for all g r o u p s are s h o w n in table 2. Patency rates w e r e a p p r o x i m a t e l y 60 p e r cent f o r n o r m a l skin, 7 p e r cent for g r a n u l a t i o n tissue a n d h y p e r t r o p h i c scars, 17 p e r cent for keloid, a n d 20 p e r cent for m a t u r e scar. Chi square analysis o f the data shown in table 2 indicated statistical significance (P < 0.001). E x a m i n i n g a 95 p e r cent c o n f i d e n c e interval for p a t e n c y across tissue g r o u p s , we c o n f i r m e d t h a t g r a n u l a t i o n tissue is similar to l l y p e r t r o p h i c scar a n d that keloid is similar to m a t u r e scar. Each g r o u p indicated a patency rate significantly lower t h a n that o f n o r m a l skin. T a b l e 3 shows the density o f endothelial cells in all microvessel profiles o b s e r v e d . A two-way analysis o f variance using the n u m b e r o f endothelial cell p r o files p e r s q u a r e m i c r o m e t e r yielded the following results: 1) patent vessels showed significantly less endothelial density t h a n n o n p a t e n t vessels (P = 0.01); 2) controlling for patency, we f o u n d no significant differences a m o n g the tissue g r o u p s (P = 0.13); a n d 3) t h e r e was no significant interaction between p a t e n c y a n d tissue g r o u p (P = 0.89).
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Analysis o f variance data for the n u m b e r s o f nuclear profiles i n d i c a t e d no significant d i f f e r e n c e s a m o n g the tissue g r o u p s studied. DISCUSSION
T i l e vast m a j o r i t y o f ttle m i c r o v e s s e l s in the h y p e r t r o p h i c scars a n d keloids had occluded lumens. T h i s f i n d i n g c o n t r a s t e d s h a r p l y with t h a t o f microvessels o b s e r v e d in n o r m a l skin. T h e fact that occluded microvessels were s o m e t i m e s o b s e r v e d in some o f o u r n o r m a l samples m a y reflect o u r inability to obtain truly n o r m a l skin samples. Most o f o u r specimens were f r o m areas ixnmediately adjacent to a lesion or were o b t a i n e d f r o m lipectomies. T h e microvessels o f the h y p e r t r o p h i c scars a n d o f m a n y keloids very often a p p e a r e d to have several satellite cells a b o u t t h e m . T h i s c o n t r a s t e d with what is usually o b s e r v e d for a capillary, w h e r e o n e perivas-
T A B L E 1.
DIAMETERS
OF MICROVESSELS
Mean __ SD
Normal skin Granulation
(n = 25)
tissue (n = 76) Hypertrophic scar (n = 306) Keloid (n = 106) Mature scar (n = 51)
(#m)
Minimum Maximum
8.2 -+ 2.7
3.3
13.9
9.2 - 2.3
4.5
13.8
8.3 - 2.3 7.8 -+ 2.4 7.7 - 2.2
3.3 3.7 3.8
14.6 14.5 13.3
MICROVESSELS AND SCAR--KlsCHER ET AL. TABLE 2.
Normal skin Granulation tissue Hypertrophic scar Keloid Mature scar
(n (n (n (n (n
= 25) = 75]') = 306) = 105i) = 51)
PATENCY OF MICROVESSELS* 95% Confidence Interval for Patency (%)z~
Patency
Partial
FuI1
(%)
Occlusion (%)
Occlusion (%)
Low
High
60 6.6 6.9 17.1 19.6
36 30.7 19.3 20.0 33.3
4 62.7 73.8 62.9 47.1
39 2 4 11 10
79 15 11 26 33
* X~ = 51.7, P < 0.001 (see text for discussion). i Data not determined for one case. :]:Documenta, Geigy. Scientific Tables, 6th Editiou, page 86, 1962. TABLE 3. No. of Vessels
Normal skin Granulation tissueqt Hypertroplfic scar Keloid Mature scar
Patent
Occludedt
15 5 21 18 10
10 70 285 87 40
ENDOTHELIAL CELL DENSITY (per/xm2) * Mean density (• Patent
0.09 0.07 0.10 0.10 0.13
_ • • • _
0.04 0.03 0.05 0.05 0.05
Minimal Density
Occluded
0.13 0.10 0.14 0.14 0.14
• • • • •
0.05 0.05 0.07 0.09 0.07
Maximal Density
Patent
Occluded
Patent
Occluded
0.05 0.04 0.03 0.03 0.08
0.07 0.03 0.03 0.03 0.05
0.15 0.11 0.19 0.24 0.26
0.23 0.29 0.53 0.52 0.33
* Results of two-way analysis of variance: effect of patency significant (P = 0.01); effect of tissue not significant (P = 0.13); no interaction between patency and tissue (P = 0.89). (See text for further discussion.) i" Includes partial and full occlusion. Data not available for one case.
cular satellite cell o r n o n e m a y be presentY. In m a n y cases the i n n e r m o s t satellite cell had every characteristic o f a myofibroblast. In any event, no previous r e p o r t k n o w n to us identifies a myofibroblast as a pericyte o f capillaries. T h e occlusion we r e p o r t may be d u e to any o f three possible mechanisms, o r combinations o f these: I) perivascular myofibroblast contraction, 2) e n d o thelial cell contraction, or 3) endothelial cell proliferation. Pericapillary myofibroblasts were o b s e r v e d in about half o f the h y p e r t r o p h i c scars a n d keloids we studied. It may be that they eventually migrate f r o m the perivascular position to the interstitial area, w h e r e they are m u c h m o r e n u m e r o u s . A f t e r a time they may be t r a n s f o r m e d into fibroblasts. Pericapillary satellite cells are suspected o f being the source o f new fibroblasts in d e r m a l w o u n d s 7-9 and, possibly, .liver cirrhosis? ~ I n d e e d , o u r first study o f g r a n u l a t i o n tissue s h o w e d a p l e t h o r a o f p e r i c y t e s a b o u t the n e w l y g r o w i n g capillaries? T h e s m o o t h c o n t o u r o f nuclear sections t h r o u g h the pericapillary myofibroblast is not c o n s i d e r e d equivocal to a p r o p o s e d active c o n t r a c tion, since only the i n n e r o r vascular side o f the cytoplasm c o n t a i n s the m i c r o f i l a m e n t s a n d densities. F u r t h e r m o r e , active contraction o f the myofibroblast, which would have a direct functional effect o n the microvessel, is suggested because the microfilaments have not been observed elsewhere in the cell. Evidence o f endothelial cell contraction, which might contribute to luminal occlusion, has not been consistently observed. C r e n a t e d nuclei are the excep-
tion, not the rule. A l t h o u g h endothelial cell contraction, as previously r e p o r t e d , n-~3 would tend to shorten a vascular segment, a bulging o f tile nuclear region could p r o t r u d e into the lumen, thereby effecting an occlusion. However, we have f o u n d no consistent evidence o f s h o r t e n e d vascular segments by multiple light microscopy studies o f serial paraffin sections. By far the most plausible mechanism for occlusion can be s u p p o r t e d by the density o f profiles o f endothelial cells observed in cross-sections. T h e endothelial cell density data also strongly a r g u e against occlusion d u e to collapse. Besides, collapse is not really tenable in light o f r o u n d profiles o f microvessels. F u r t h e r m o r e , figure 2, in which a microvessel 8/~m in d i a m e t e r is occluded, showing profiles o f 12 e n d o thelial cells, also d e m o n s t r a t e s tight endothelial j u n c tions situated high between endothelial cells. This was a c o m m o n observation; it would not o c c u r if the endothelial cell density increased at a given focus o f the vessel owing to either contraction or endothelial cell collapse. It also is not reasonable to suspect that tim occluded microvessels in h y p e r t r o p h i c scar a n d keloid are simply the terminal closed ends o f g r o w i n g vessel buds. I n h y p e r t r o p h i c scars a n d keloids o f s o m e years' standing, this activity, expected in g r a n u l a t i o n tissue, would have long since ceased. Occlusion is essentially the same process in each g r o u p , as j u d g e d f r o m the interaction statistic. T h e density data also suggest that some n o r m a l cases were probably not normal. I n d e e d , some o f those cases were probably " n o r m a l " tissue immediately adjacent to the scar or keloid lesion.
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H U M A N P A T H O L O G Y - - V O L U M E 13, NUMBER 9
September1982
It is likely that e n d o t h e l i a l cell p r o l i f e r a t i o n is t h e m a j o r f a c t o r c o n t r i b u t i n g to occlusion. H o w e v e r , it is also likely t h a t p e r i v a s c u l a r m y o f i b r o b l a s t a n d e n d o thelial cell c o n t r a c t i o n s also c o n t r i b u t e , albeit minimally. T h e u l t r a s t r u c t u r e o f m o s t e n d o t h e l i a l cells in h y p e r t r o p h i c scar a n d keloid is in c o n c e r t with an active biologic state. W h e t h e r this indicates p r o l i f e r a tion c a n n o t be definitely stated here. H o w e v e r , p r o liferation c o u l d be s u p p o r t e d by i n c r e a s e d n u m b e r s o f o r g a n e l l e s a n d m a n y intercellular j u n c t i o n s t h a t f o r m c o m p l e x u n i o n s o r m a z e s o f intricate partially i n t e r d i g i t a t e d cell processes. It is o f special significance that in o u r p r e v i o u s s t u d y o f a first series o f g r a n u l a t i o n tissues f r o m d e e p h u m a n w o u n d s a we f o u n d o c c l u d e d microvessels o n l y in o l d e r g r a n u l a t i o n s , t h o s e in w h i c h t h e c o l l a g e n patterns of hypertrophic nodules had already been well established. A d d i t i o n a l l y , fibrin p o l y m e r was persistent in e v e r y case e x a m i n e d . It a p p e a r s likely t h a t m i c r o v a s c u l a r o c c l u s i o n in h y p e r t r o p h i c scars a n d keloids is d u e principally to i n c r e a s e d n u m b e r s o f e n d o t h e l i a l cells. It m a y also be t h a t a p r o l i f e r a t i o n is established in t h e g r a n u l a t i o n phase. T h e factors p r o m o t i n g a s u s p e c t e d p r o l i f e r a tion are n o t k n o w n ; h o w e v e r , b e c a u s e we h a v e obs e r v e d pervasive a n d persistent fibrin in o u r series o f g r a n u l a t i o n tissues, we s u g g e s t that this is a possible s o u r c e o f stimulation. Fibrin has b e e n i d e n t i f i e d as stimulating a marked uptake ofSH-labeled thymidine in c u l t u r e s o f fibroblasts, t4"t5 O n c e again, we a r e c o m p e l l e d to r e a f f i r m o u r h y p t h e s i s t h a t h y p o x i a is an i n t e g r a l f a c t o r in t h e g e n e r a t i o n o f h y p e r t r o p h i c scar a n d keloid. W e h a v e provided strong morphologic evidence of the origin o f t h e h y p o x i a . W h e t h e r the h y p o x i a m a y stimulate p r o l i f e r a t i o n o f e n d o t h e l i a l cells, pericytes, a n d fibroblasts o r the synthesis o f collagen, o r all, r e m a i n s to be d e t e r m i n e d . H u n t et al. ~6 a r g u e c o n v i n c i n g l y f o r
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h y p o x i a c o n t r o l l i n g collagen synthesis in the w o u n d s o f rabbits a n d dogs.
REFERENCES I. Kischer CW, Linares H, Dobrkovsky M, et al: Electron microscop)' of the hypertrophic scar. Twenty-ninth Annual Proceedings of the Society of America, 1971, p 302. 2. Kischer CW, Shetlar MR, Shet[ar CL: Alterations of hypertrophic scars induced by mechanical pressure. Arch Dermatol I11:60, 197.6 3. Kischer CW: Fine structure of granulation tissues from deep injury. J Invest Dermatol 72:147, 1979 4. Kischer CW, Shetlar MR: Microvasculature in hypertrophic scars and the effects of pressure. J Trauma 19:757, 1979 5. Sloan DF, Brown RD, Wells CH: Tissue gases in human hypertrophic burn scars. Plast Reconstr Surg 61:431, 1978 6. CliffWJ: Blood Vessels. London, Cambridge University Press, 1976, pp 68-72 7. Weber K, Braun-Falco O: Ultrastructure of blood vessels in human granulation tissue. Arch Dermatol Fortschr 248:29, 1973 8. Crocker DJ, Murad TM, Greer JC: Role of the pericyte in wound healing: an uhrastructural study. Exp Mol Pathol 13:51, 1970 9. Ross R, Everett NB, Tyler R: Wound healing and collagen formation. VI. The origin ofthe wound fibroblast studied in parabiosis. J Cell Biol 44:645, 1970 10. hie C, Kocher O, Gabbiani G: Contractility of myofibroblasts during experimental liver cirrhosis. J Submicrosc Cytol 12:209, 1980 11. Becker CG, Nachman RL: Contractile proteins of endothelial cells, platelets anti smooth muscle. Am J Pathol 71:1, 1973 12. Cliff, Wj: Blood Vessels. London, Cambridge University Press, 1976, pp 154-155 13. Majno G, Shea SM, Leventhal M: Endothelial contraction induced by histamine-type mediators.J Ceil Bio142:647, 1969 14. Nozawa RT: Altered morphology and increased cell adhesiveness of Chinese hamster ovary cells cultured on fibrin. J Cell Physiol 90:351, 1977. 15. Kittlick PD: Fibrin in fibroblast cultures: a metabolic study as a contribution to inflammation and tissue repair. Exp Pathol 17:312, 1979 16. Hunt TK, ConoIly WB, Aronson SB, et al: Anaerobic metabolism and wound healing: an hypothesis for the initiation and cessation of collagen synthesis in wounds. Am J Surg 135:328, 1979