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Loss of factor XIIIa in pig dermis during tissue expansion
Journal of Dermatological
Science, 2 (1991)
62
62-65
Elsevier
DESC 00062
Loss of factor XIIIa in pig dermis during tissue expansion * Neal S. Penneys, Department
of Dermatology.
‘B. Rademaker,
’
I.T. Jackson
and
’
M.C. Fasching
Universitv of Miami School of Medicine, Miami. Florida, and the Reconstructive Surgery, Mayo Clinic, Rochester, Minnesota,
(Received 24 March; accepted
Key words: Factor XIII; Fibroplasia;
1 October
’ Department of Plastic and
U.S.A.
1990)
Tissue expansion;
Inhibition
Abstract
Considerable evidence suggests that factor XIIIa, a blood coagulation factor, also functions in tissue repair, and specifically in the dynamic process of fibroplasia. On the other hand, the dynamic process of tissue expansion would require a loosening of the dermis, the opposite of fibroplasia. We examined the effects of tissue expansion on the expression of factor XIIIa in pig skin. Standard immunohistochemical procedures were used coupled with a rabbit antibody to factor XIIIa. In this report, we demonstrate that the normal expression of factor XIIIa in pig dermis is completely inhibited at the apex overlying the tissue expander. Factor XIIIa inhibition during expansion supports the thesis that this substance is important in the production and maintenance of tibroplasia.
Introduction Factor XIII, a blood proenzyme distributed in plasma and platelets, is part of the final common blood coagulation pathway where its function is to stabilize clots by crosslinking fibrin to protein. Two forms of the transglutaminase exist: extracellular or plasma factor XIII and an intracellular factor XIII. The extracellular factor XIII is a tetramer consisting of a dimer of the active ‘a’ subunit attached to a carrier protein dimer. The intracellular factor XIIIa is a dimer of ‘a’ subunits only. The ‘a’ subunits in both forms are identical [ 11. Intracellular factor XIIIa has been identified in many tissues including platelets, megakaryocytes [2,3], placenta, uterus, prostate tissue [4,5], Correspondence to: N.S. Penneys, Department of Dermatology, University ofMiami School of Medicine, Box 016940, Miami, FL 33101, U.S.A.
0923-181 l/91/$03.50
0 1991 Elsevier Science Publishers
human peripheral blood monocytes, peritoneal macrophages [ 61, alveolar macrophages, U937 cells (a human promonocytic tumor line) /7], fibroblast-like mesenchymal cells [S], and in human dendritic reticulum cells in reactive lymphoid follicles [ 91.
Fig. 1. Tissue expanders are in place and inflated dorsum of a pig.
B.V. (Biomedical
Division)
on the
63
Iwabuchi [lo], while investigating the postulated site of factor XIII synthesis in the liver, found that portal tibroblasts in livers damaged by alcoholic cirrhosis and chronic active hepatitis stained with both factor XIII subunits. These findings were confirmed by others [ 111, who identified factor XIII in reactive fibroblasts, among other cell types. Factor XIIIa and Factor XIIIs have been found in reactive and neoplastic fibroblastic and fibrohistiocytic lesions [ 121. Factor XIIIa has not been found in granulocytes, lymphocytes [ 681, epithelial cells, endothelial cells, or mast cells [8]. Tissue expanders stretch normal skin prior to a variety of surgical procedures. Little is known of the biochemical alterations in the skin which follows expansion of this tissue. The cross-linked product of the reaction catalyzed by factor XIIIa should make tissue expansion more difficult. Consequently, we hypothesized that tissue expansion would inhibit tissue factor XIIIa expression.
LaJolla, CA) [ 131. Appropriate positive and negative controls were included in each experiment. Other antibodies used on human tissues such as vimentin, KP-1 and lysozyme did not routinely label formalin-fixed par&m embedded sections of pig skin. One thousand papillary dermal cells not identifiable as endothelial cells were examined from each area and the percentage of cells expressing factor XIIIa determined. Results
Rabbit antibody against human factor XIIIa labeled 720 of 1,000 (72%) cells in the papillary dermis of normal pig skin (Fig. 2) but few in the reticular dermis. After expansion, factor XIIIa was found in only 82 of 1,000 (8.2%) cells in the papillary dermis at the apex overlying the tissue
Methods
Four swine weighing approximately 20 kg each received 4 expanders placed subcutaneously under the skin overlying the back; 2 on each side (Fig. 1). Each Radovan-type expander measured 5 by 9 cm with a listed maximum volume of 450 ml. The skin was shaved with an electric clipper over an area leaving a margin of 10 cm around each expander. The tissue expanders were placed 3 to 5 cm below the dorsum of the pig, two on each side (one cephalad, the other, caudad). Ports for inflation were placed subcutaneously. The expanders were inflated with saline 7 days after surgery and then each day for 7 days thereafter. At the end of this period, biopsy samples were removed and fixed in buffered formaldehyde solution. Expansion pressures were 50 mmHg, 75 mmHg, 150 mmHg, and 200 mmHg. The immunohistochemical demonstration of factor XIIIa was performed using standard techniques and the avidin-biotin-peroxidase complex system with a polyclonal rabbit antibody against human factor XIIIa (Behring Diagnostics,
Fig. 2. Immunostaining for factor XIIIa in normal pig skin. Othe@han endothelial cells, almost all papillary dermal cells are expressing factor XIIIa in this field. (all figures, oxidized diaminobenzidine and hematoxylin) ( x 200).
64
Fig. 3. There is no evidence of binding of antibody to factor XIIIa to papillary dermal cells at the apex of the tissue expander. ( x 400).
Fig. 4. An occasional dermal cell contains factor XIIIa at the margin of the tissue expander. ( x 200).
expander (Fig. 3) and diminished expression (relative to that of normal skin) of factor XIIIa was noted at the margin of the expander (433 of 1,000 cells examined; 43.3%) (Fig. 4). Similar changes were observed for the 4 different expansion pressures. Similar cell densities were found in the papillary dermis at the apex of the expander when compared to that at the periphery in normal skin; decreased expression of factor XIIIa does not appear to be a dilutional effect. In cells that were expressing factor XIIIa at the apex and margins of the expander, the intensity and granularity of the binding reaction was less than that in normal skin.
tissue expansion are many: 1) tissue is available from a local site, without involving transfer over any distance; 2) excellent color match and texture result because the tissue is expanded in an area adjacent to the defect; 3) sensation is maintained; and 4) there is no donor site requiring surgical repair. Morphologically, the epidermis responds to tissue expansion by significantly increasing mitotic activity [ 141. Interestingly, the epidermis does not thin during the process of expansion [ 151. The dermis and subcutis on the other hand thin signilicantly after expansion. A capsule forms adjacent to the expander. Extracellular changes have been described in expanded skin. New collagen fibrils of variable diameter and increased numbers of fibroblasts with active organelles occur in this tissue. Myotibroblasts were found in deeper parts
Tissue expansion is an important surgical technique for repairing defects. The advantages of
65
of the expanded dermis [ 151. Endothelial cells in capillaries possessed large numbers of WeibelPalade bodies, ribosomes and glycogen-like particles [ 151. In man, all changes revert to normal by 2 years after expansion [ 151. There is no significant difference in epidermal and dermal thickness in relation to expansion time, expander volume and location of the expander or the age of the patient with the implant. Little is known of the biochemical processes which follow tissue expansion. Mediators must be synthesized and released which stimulate epidermal proliferation, dermal fibroplasia, and vascular proliferation. In this study, we have demonstrated that factor XIIIa, a transglutaminase which is present in many of the papillary dermal cells of normal pig skin, is absent in papillary dermis at the apex of the tissue expander and markedly decreased in papillary dermal cells at the margins of the expander. We hypothesize that the absence of factor XIIIa in these cells represents either the loss of a cytokine that normally stimulates cells in this region to synthesize factor XIIIa or the release of an inhibitor of factor XIIIa synthesis from a local site. Bone marrow-derived cells can contain factor XIIIa; based on this observation, another possibility is that expansion inhibits the egress of factor XIIIa-positive cells into the dermis at the apex of the expander. In other work, we have shown that factor XIIIa expression in human skin is related to processes characterized by fibroplasia and we hypothesize that factor XIIIa has an important, but as yet undefined role, in the general process of fibroplasia [ 161. The stretch stimulus is in many ways opposite to that which results in fibroplasia; in this context, it is logical that factor XIIIa, known to be expressed in fibroplasia, would be inhibited in skin undergoing tissue expansion. References 1 Schwartz ML, Pizzo SV, Hill RL, McKee PA: Human factor XIII from plasma and platelets: Molecular weights, subunit structure, proteolytic activation and cross linking of fibrinogen and fibrin. J Biol Chem 248: 1395-1401. 1973.
2 Kiesselbach TH, Wagner RH: Demonstration of factor XIII in human megakaryocytes by a fluorescent antibody technique. Ann NY Acad Sci 202: 318-28, 1972. 3 Rider DM, McDonagh RP, McDonagh J: A possible contributory role of the platelet in the formation of plasma factor XIII. Br J Haematol 39: 579-88, 1978. 4 Chung SI: Comparative studies on tissue transglutaminase and factor XIII. Ann NY Acad Sci 202: 240-55, 1972. 5 Bohn H: Comparative studies on the fibrin-stabilizing factors from human plasma, platelets, and placentas. Ann NY Acad Sci 202: 256-72, 1972. 6 Henriksson P, Becker S, Lynch G, McDonagh J: Identification of intracellular factor XIII in human monocytes and macrophages. J Clin Invest 76: 528-34, 1985. 7 Kradin RL, Lynch GW, Kurmick JT, Erikson M, Colvin RB, McDonagh J: Factor XIIIa is synthesized and expressed on the surface of U937 cells and alveolar macrophages. Blood 69: 778-85, 1987. 8 Nemes Z, Thomazy V, Adany R, Muszbek L: Identilication of histiocytic reticulum cells by the immunohistochemical demonstration of factor XIII (F-XIIIa) in human lymph nodes. J Path01 149: 121-32, 1986. 9 Nemes Z, Adany R, Thomazy J: Selective visualization of human dendritic reticulum cells in reactive lymphoid follicles by the immunohistochemical demonstration of the subunit A of factor XIII (F-XIIIa). Virchows Arch B 52: 453-6, 1987. 10 Iwabuchi S: Hepatic fibrosis and coagulation factor XIII. Jpn J Gastroenterol 80: 2229-39, 1983. 11 Fear JD, Jackson P, Gray C, Miloszewski K, Losowsky M: Localization of factor XIII in human tissue using immunoperoxidase technique. J Clin Pathol 37: 560-3, 1984. 12 Reid MB, Gray C, Fear JD, Bird CC: Immunohistochemical demonstration of factors XIIIa and XIIIs in reactive and neoplastic fibroblastic and librohistiocytic lesions. Histopathology 10: 1171-8, 1986. 13 Nemeth AJ, Penneys NS, Bernstein HB: Fibrous papule: A tumor offibrohistiocytic cells that contain factor XIIIa. J Am Acad Dermatol 19: 1102-l 106, 1988. 14 Francis AJ, Marks R: Skin stretching and epidermopoiesis. Br J Exp Path01 58: 35-39, 1977. 15 Pasyk KA, Argenta LC, Austad ED: Histopathology of human expanded tissue. Clinics Plastic Surg 14: 435-445, 1987. 16 Penneys NS: Factor XIII expression in the skin: Observations and a hypothesis. J Am Acad Dermatol 22: 484-8, 1990.
Science, 2 (1991)
62
62-65
Elsevier
DESC 00062
Loss of factor XIIIa in pig dermis during tissue expansion * Neal S. Penneys, Department
of Dermatology.
‘B. Rademaker,
’
I.T. Jackson
and
’
M.C. Fasching
Universitv of Miami School of Medicine, Miami. Florida, and the Reconstructive Surgery, Mayo Clinic, Rochester, Minnesota,
(Received 24 March; accepted
Key words: Factor XIII; Fibroplasia;
1 October
’ Department of Plastic and
U.S.A.
1990)
Tissue expansion;
Inhibition
Abstract
Considerable evidence suggests that factor XIIIa, a blood coagulation factor, also functions in tissue repair, and specifically in the dynamic process of fibroplasia. On the other hand, the dynamic process of tissue expansion would require a loosening of the dermis, the opposite of fibroplasia. We examined the effects of tissue expansion on the expression of factor XIIIa in pig skin. Standard immunohistochemical procedures were used coupled with a rabbit antibody to factor XIIIa. In this report, we demonstrate that the normal expression of factor XIIIa in pig dermis is completely inhibited at the apex overlying the tissue expander. Factor XIIIa inhibition during expansion supports the thesis that this substance is important in the production and maintenance of tibroplasia.
Introduction Factor XIII, a blood proenzyme distributed in plasma and platelets, is part of the final common blood coagulation pathway where its function is to stabilize clots by crosslinking fibrin to protein. Two forms of the transglutaminase exist: extracellular or plasma factor XIII and an intracellular factor XIII. The extracellular factor XIII is a tetramer consisting of a dimer of the active ‘a’ subunit attached to a carrier protein dimer. The intracellular factor XIIIa is a dimer of ‘a’ subunits only. The ‘a’ subunits in both forms are identical [ 11. Intracellular factor XIIIa has been identified in many tissues including platelets, megakaryocytes [2,3], placenta, uterus, prostate tissue [4,5], Correspondence to: N.S. Penneys, Department of Dermatology, University ofMiami School of Medicine, Box 016940, Miami, FL 33101, U.S.A.
0923-181 l/91/$03.50
0 1991 Elsevier Science Publishers
human peripheral blood monocytes, peritoneal macrophages [ 61, alveolar macrophages, U937 cells (a human promonocytic tumor line) /7], fibroblast-like mesenchymal cells [S], and in human dendritic reticulum cells in reactive lymphoid follicles [ 91.
Fig. 1. Tissue expanders are in place and inflated dorsum of a pig.
B.V. (Biomedical
Division)
on the
63
Iwabuchi [lo], while investigating the postulated site of factor XIII synthesis in the liver, found that portal tibroblasts in livers damaged by alcoholic cirrhosis and chronic active hepatitis stained with both factor XIII subunits. These findings were confirmed by others [ 111, who identified factor XIII in reactive fibroblasts, among other cell types. Factor XIIIa and Factor XIIIs have been found in reactive and neoplastic fibroblastic and fibrohistiocytic lesions [ 121. Factor XIIIa has not been found in granulocytes, lymphocytes [ 681, epithelial cells, endothelial cells, or mast cells [8]. Tissue expanders stretch normal skin prior to a variety of surgical procedures. Little is known of the biochemical alterations in the skin which follows expansion of this tissue. The cross-linked product of the reaction catalyzed by factor XIIIa should make tissue expansion more difficult. Consequently, we hypothesized that tissue expansion would inhibit tissue factor XIIIa expression.
LaJolla, CA) [ 131. Appropriate positive and negative controls were included in each experiment. Other antibodies used on human tissues such as vimentin, KP-1 and lysozyme did not routinely label formalin-fixed par&m embedded sections of pig skin. One thousand papillary dermal cells not identifiable as endothelial cells were examined from each area and the percentage of cells expressing factor XIIIa determined. Results
Rabbit antibody against human factor XIIIa labeled 720 of 1,000 (72%) cells in the papillary dermis of normal pig skin (Fig. 2) but few in the reticular dermis. After expansion, factor XIIIa was found in only 82 of 1,000 (8.2%) cells in the papillary dermis at the apex overlying the tissue
Methods
Four swine weighing approximately 20 kg each received 4 expanders placed subcutaneously under the skin overlying the back; 2 on each side (Fig. 1). Each Radovan-type expander measured 5 by 9 cm with a listed maximum volume of 450 ml. The skin was shaved with an electric clipper over an area leaving a margin of 10 cm around each expander. The tissue expanders were placed 3 to 5 cm below the dorsum of the pig, two on each side (one cephalad, the other, caudad). Ports for inflation were placed subcutaneously. The expanders were inflated with saline 7 days after surgery and then each day for 7 days thereafter. At the end of this period, biopsy samples were removed and fixed in buffered formaldehyde solution. Expansion pressures were 50 mmHg, 75 mmHg, 150 mmHg, and 200 mmHg. The immunohistochemical demonstration of factor XIIIa was performed using standard techniques and the avidin-biotin-peroxidase complex system with a polyclonal rabbit antibody against human factor XIIIa (Behring Diagnostics,
Fig. 2. Immunostaining for factor XIIIa in normal pig skin. Othe@han endothelial cells, almost all papillary dermal cells are expressing factor XIIIa in this field. (all figures, oxidized diaminobenzidine and hematoxylin) ( x 200).
64
Fig. 3. There is no evidence of binding of antibody to factor XIIIa to papillary dermal cells at the apex of the tissue expander. ( x 400).
Fig. 4. An occasional dermal cell contains factor XIIIa at the margin of the tissue expander. ( x 200).
expander (Fig. 3) and diminished expression (relative to that of normal skin) of factor XIIIa was noted at the margin of the expander (433 of 1,000 cells examined; 43.3%) (Fig. 4). Similar changes were observed for the 4 different expansion pressures. Similar cell densities were found in the papillary dermis at the apex of the expander when compared to that at the periphery in normal skin; decreased expression of factor XIIIa does not appear to be a dilutional effect. In cells that were expressing factor XIIIa at the apex and margins of the expander, the intensity and granularity of the binding reaction was less than that in normal skin.
tissue expansion are many: 1) tissue is available from a local site, without involving transfer over any distance; 2) excellent color match and texture result because the tissue is expanded in an area adjacent to the defect; 3) sensation is maintained; and 4) there is no donor site requiring surgical repair. Morphologically, the epidermis responds to tissue expansion by significantly increasing mitotic activity [ 141. Interestingly, the epidermis does not thin during the process of expansion [ 151. The dermis and subcutis on the other hand thin signilicantly after expansion. A capsule forms adjacent to the expander. Extracellular changes have been described in expanded skin. New collagen fibrils of variable diameter and increased numbers of fibroblasts with active organelles occur in this tissue. Myotibroblasts were found in deeper parts
Tissue expansion is an important surgical technique for repairing defects. The advantages of
65
of the expanded dermis [ 151. Endothelial cells in capillaries possessed large numbers of WeibelPalade bodies, ribosomes and glycogen-like particles [ 151. In man, all changes revert to normal by 2 years after expansion [ 151. There is no significant difference in epidermal and dermal thickness in relation to expansion time, expander volume and location of the expander or the age of the patient with the implant. Little is known of the biochemical processes which follow tissue expansion. Mediators must be synthesized and released which stimulate epidermal proliferation, dermal fibroplasia, and vascular proliferation. In this study, we have demonstrated that factor XIIIa, a transglutaminase which is present in many of the papillary dermal cells of normal pig skin, is absent in papillary dermis at the apex of the tissue expander and markedly decreased in papillary dermal cells at the margins of the expander. We hypothesize that the absence of factor XIIIa in these cells represents either the loss of a cytokine that normally stimulates cells in this region to synthesize factor XIIIa or the release of an inhibitor of factor XIIIa synthesis from a local site. Bone marrow-derived cells can contain factor XIIIa; based on this observation, another possibility is that expansion inhibits the egress of factor XIIIa-positive cells into the dermis at the apex of the expander. In other work, we have shown that factor XIIIa expression in human skin is related to processes characterized by fibroplasia and we hypothesize that factor XIIIa has an important, but as yet undefined role, in the general process of fibroplasia [ 161. The stretch stimulus is in many ways opposite to that which results in fibroplasia; in this context, it is logical that factor XIIIa, known to be expressed in fibroplasia, would be inhibited in skin undergoing tissue expansion. References 1 Schwartz ML, Pizzo SV, Hill RL, McKee PA: Human factor XIII from plasma and platelets: Molecular weights, subunit structure, proteolytic activation and cross linking of fibrinogen and fibrin. J Biol Chem 248: 1395-1401. 1973.
2 Kiesselbach TH, Wagner RH: Demonstration of factor XIII in human megakaryocytes by a fluorescent antibody technique. Ann NY Acad Sci 202: 318-28, 1972. 3 Rider DM, McDonagh RP, McDonagh J: A possible contributory role of the platelet in the formation of plasma factor XIII. Br J Haematol 39: 579-88, 1978. 4 Chung SI: Comparative studies on tissue transglutaminase and factor XIII. Ann NY Acad Sci 202: 240-55, 1972. 5 Bohn H: Comparative studies on the fibrin-stabilizing factors from human plasma, platelets, and placentas. Ann NY Acad Sci 202: 256-72, 1972. 6 Henriksson P, Becker S, Lynch G, McDonagh J: Identification of intracellular factor XIII in human monocytes and macrophages. J Clin Invest 76: 528-34, 1985. 7 Kradin RL, Lynch GW, Kurmick JT, Erikson M, Colvin RB, McDonagh J: Factor XIIIa is synthesized and expressed on the surface of U937 cells and alveolar macrophages. Blood 69: 778-85, 1987. 8 Nemes Z, Thomazy V, Adany R, Muszbek L: Identilication of histiocytic reticulum cells by the immunohistochemical demonstration of factor XIII (F-XIIIa) in human lymph nodes. J Path01 149: 121-32, 1986. 9 Nemes Z, Adany R, Thomazy J: Selective visualization of human dendritic reticulum cells in reactive lymphoid follicles by the immunohistochemical demonstration of the subunit A of factor XIII (F-XIIIa). Virchows Arch B 52: 453-6, 1987. 10 Iwabuchi S: Hepatic fibrosis and coagulation factor XIII. Jpn J Gastroenterol 80: 2229-39, 1983. 11 Fear JD, Jackson P, Gray C, Miloszewski K, Losowsky M: Localization of factor XIII in human tissue using immunoperoxidase technique. J Clin Pathol 37: 560-3, 1984. 12 Reid MB, Gray C, Fear JD, Bird CC: Immunohistochemical demonstration of factors XIIIa and XIIIs in reactive and neoplastic fibroblastic and librohistiocytic lesions. Histopathology 10: 1171-8, 1986. 13 Nemeth AJ, Penneys NS, Bernstein HB: Fibrous papule: A tumor offibrohistiocytic cells that contain factor XIIIa. J Am Acad Dermatol 19: 1102-l 106, 1988. 14 Francis AJ, Marks R: Skin stretching and epidermopoiesis. Br J Exp Path01 58: 35-39, 1977. 15 Pasyk KA, Argenta LC, Austad ED: Histopathology of human expanded tissue. Clinics Plastic Surg 14: 435-445, 1987. 16 Penneys NS: Factor XIII expression in the skin: Observations and a hypothesis. J Am Acad Dermatol 22: 484-8, 1990.