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Localization and Regulation of Pregnancy-Associated Plasma Protein A Expression in Healing Human Skin

Endocrine Research Unit, Mayo Clinic and Foundation (B.-K.C., C.A.C.), Rochester, Minnesota 55905; Department of Dermatology, University of Utah Health Sciences Center (K.M.L.), Salt Lake City, Utah 84132; Department of Dermatology, Mayo Clinic and Mayo Foundation (M.R.P.), Rochester, Minnesota 55905; and University of Aarhus (M.T.O., C.O.), DK-8000 Aarhus C, Denmark

Address all correspondence and requests for reprints to: Cheryl A. Conover, Ph.D., Mayo Clinic, 200 First Street SW, 5-194 Joseph, Rochester, Minnesota 55905. E-mail: conover.cheryl{at}mayo.edu.

	Abstract

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Pregnancy-associated plasma protein A (PAPP-A) is an IGF-binding protein-4 (IGFBP-4) metalloproteinase that cleaves inhibitory IGFBP-4 to amplify local IGF-I bioavailability in vitro. Thus it has functional implications in injury/repair responses. In this study we determined PAPP-A expression in healing human skin. Wounds were induced with a scalpel on the forearms of three normal subjects and were allowed to heal by first intention. Biopsies obtained on d 0, 2, 8, and 14 were processed for immunohistochemical detection of PAPP-A, IGF-I, and IGFBP-4. In uninjured skin (d 0), strong staining for PAPP-A was present in the epidermis, sweat and sebaceous gland epithelial cells, hair follicles, and blood vessels; no PAPP-A was detected in dermal fibroblasts or with mature collagen bundles. IGF-I localized strongly to epithelial cells of skin glands was weak to moderate in epidermis and blood vessels, and was absent in dermal cells. Weak focal staining for IGFBP-4 was found within uninjured epidermis. During wound healing, PAPP-A expression was induced in dermal granulation tissue within and adjacent to the injury. PAPP-A was present in dermis on d 2 and was increased in intensity and extent on d 8 and 14. PAPP-A expression also increased in the epidermis. PAPP-A expression in cells of granulation tissue colocalized with -smooth actin staining of myofibroblasts and new blood vessels as well as with CD68 staining of macrophages and was associated with the compact, newly synthesized collagen of the healing wound. IGF-I staining was enhanced in the epidermis localized to the area of the incision and in granulation tissue associated with lymphoid cells. IGFBP-4 staining of the epidermis remained unchanged during wound healing, but was induced in the fibroblastic cells of granulation tissue over time. These data demonstrate localized and regulated expression of PAPP-A in human skin and suggest that PAPP-A may play an important role in an integrated IGF system in wound healing and tissue remodeling in vivo.

	Introduction

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CUTANEOUS WOUND HEALING is a complex biological process requiring growth factors produced at the wound site that act in concert to restore the integrity of the injured tissue (reviewed in Refs. 1, 2, 3, 4, 5). There is substantive evidence that IGF-I is one of the most important growth factors in wound healing. IGF-I is present throughout the healing process in skin wounds of humans and various animals (6, 7, 8, 9, 10). IGFs released at the wound site are presumed to be a necessary part of the natural healing machinery. Several cell types involved in the wound process, such as macrophages and platelet -granules, secrete IGF-I (11, 12, 13). Epidermal keratinocytes and dermal fibroblasts possess IGF-I receptors and are responsive to IGF-I. IGF-I stimulates keratinocyte and fibroblast proliferation and matrix protein synthesis in vitro (14, 15, 16), and exogenous IGF-I in combination with other growth factors, such as platelet-derived growth factor, has been shown to accelerate wound healing in vivo (17).

IGF-I action is regulated by a family of six IGF-binding proteins (IGFBPs) (18, 19). Batch et al. (20) demonstrated the expression of IGFBP-2, -3, -4, and -5 in human skin by in situ hybridization. Human fibroblasts in culture produce IGFBP-3, -4, and -5 (21). Keratinocytes predominantly produce IGFBP-2, -3, and -4 (22). Thus, secreted IGF-I in wound sites is presumably complexed to IGFBPs and unavailable for receptor interaction. It is postulated that IGFBPs undergo limited proteolysis to deliver bioactive IGF-I and stimulate new growth at the wound site. IGFBPs can serve as substrate for several established proteases, such as cathepsin, plasmin, and thrombin (23, 24). Moreover, a specific metalloproteinase secreted by cultured human skin fibroblasts that cleaves IGFBP-4 in vitro was recently identified as pregnancy-associated plasma protein A (PAPP-A) (25). Because of its ability to degrade an inhibitory IGFBP, PAPP-A has the potential to amplify local IGF-I activity (26, 27). Recent revelations that PAPP-A expression is increased in vascular repair and inflammation point to new role for PAPP-A in the injury/repair response (28, 29). In this study we determined PAPP-A expression in healing human skin in vivo and the coordinate expression of IGF-I and IGFBP-4.

	Materials and Methods

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Wounding and tissue specimens

First intention wounds were induced with a scalpel on the volar aspect of each forearm of three normal adult subjects after local anesthesia with 2% xylocaine, producing a 1-cm long incision extending into the dermis. Four-millimeter punch biopsy specimens were obtained on d 0, 2, 8, and 14 following wounding, and were fixed in 10% buffered formalin and embedded in paraffin. Five-micrometer thick serial sections were mounted on polylysine-coated slides (SuperFrost Plus, Fisher Scientific, Pittsburgh, PA) using a 2035 Jung Biocut microtome (Leica Instruments, Nussloch, Germany). This study was approved by the Mayo Clinic institutional review board, and volunteers gave written consent for the biopsies.

Immunohistochemistry

Immunohistochemistry was performed using specific monoclonal antibodies against PAPP-A [monoclonal antibody 234-5 (29, 30), final concentration, 10 µg/ml], IGF-I (provided by Diagnostic Systems Laboratories, Inc., Webster, TX; 2.5 µg/ml), IGFBP-4 (Austral Biologicals, San Ramon, CA; 10 µg/ml), -smooth muscle actin (-SMA; DAKO, Glostrup, Denmark; 0.9 µg/ml), and macrophage marker, CD68 (DAKO; 4 µg/ml).

Sections were dewaxed, rehydrated, steamed with citric acid, and then incubated in methanol containing 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. After washing with Tris-buffered saline (pH 7.4), the sections were reacted with Protein Block (DAKO) for 20 min to block nonspecific binding of the antibodies, and then incubated with primary antibodies in a humidified chamber at room temperature for 1 h. Sections were rinsed three times with Tris-buffered saline and then incubated with secondary antibody (Envision Plus Systems, DAKO) for 30 min at room temperature. After rinsing three times, Nova Red substrate (Vector Laboratories, Inc., Burlingame, CA) was added at room temperature for 2 min to produce a red reaction product. All sections were counterstained with Gill’s hematoxylin, dehydrated, mounted, and visualized by light microscopy. Positive and negative control slides were processed in parallel. For the negative control, monoclonal antibodies were replaced with monoclonal mouse immunoglobulin (IgG; DAKO) at the appropriate concentration.

Coded slides were evaluated by two investigators (B.-K.C. and C.A.C.) independently and were graded as negative (-), weak (+), moderate (++), or strong (+++), referring to the intensity of immunostaining for each antibody. The individual scores of the reviewers as well as the results from the three subjects at each time point (revealed after breaking the code) were in concordance and, therefore, are presented as a single score in the tables. A third investigator (M.R.P.) reviewed the evaluation results. Images of the stained sections were captured using a microscope (Microphot-FXA, Nikon, Garden City, NJ) equipped with a camera (Zeiss Progres 3012, Jenoptik, Jena, Germany) and assembled using Photoshop 5 (Adobe Systems, Inc., San Jose, CA).

	Results

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Intact tissue

Adult skin consists of two main tissue layers: keratinized stratified epidermis and collagen-rich dermal connective tissue. Appendages such as hair follicles and glands are primarily epidermal derived, but project into the dermal layer. In unwounded adult human skin, strong staining for PAPP-A was observed in the epidermis, sweat and sebaceous gland epithelial cells, hair follicles, and endothelial cells of capillaries. There was no detectable PAPP-A staining of the wavy bundles of mature collagen in the dermal sheath (Fig. 1A, Fig. 3A, and Table 1). For IGF-I (Fig. 1B and Table 1), intense staining was found in the epithelial cells of sweat gland coils, sebaceous glands, and hair follicles. The staining intensity of the sweat glands was variable, with the most intense staining associated with the luminal or secretory cells. This suggests differential IGF-I expression depending on the differentiation stage of the glandular epithelial cells. Moderate staining for IGF-I was present in skin smooth muscle (musculi arrectores pili), and weak staining was seen in the epidermis and blood vessels. The pattern of staining for IGFBP-4 was similar to that of IGF-I; however, the staining intensity was much weaker and more variable. In addition, there appeared to be clusters of relatively intense staining for IGFBP-4 in cells nested in the epidermis (Fig. 1C and Table 1). Some staining for IGFBP-4 could be detected in the dermis associated with the occasional fibroblast. Negative staining with nonspecific mouse IgG is shown in Fig. 1D.

View larger version (119K): [in this window] [in a new window]   FIG. 1. Unwounded human skin: immunostaining for PAPP-A (A), IGF-I (B), and IGFBP-4 (C). D, Negative staining with nonspecific mouse IgG. Magnification, x60. e, Epidermis; sw, sweat gland; bv, blood vessel.

View larger version (144K): [in this window] [in a new window]   FIG. 3. Immunostaining of myofibroblasts in granulation tissue for PAPP-A (A and C) and -SMA (B and D) on uninjured (A and B) and d 8 healing (C and D) human skin. PAPP-A staining is associated with activated fibroblasts (myofibroblasts, indicated by arrows), a cell type that also stains positively for -SMA. Magnification, x3000.

Healing of a skin-wounding injury is a dynamic, interactive repair process with overlapping phases of hemostasis inflammation, epidermal and dermal tissue reformation, and tissue remodeling in a defined timeline (1, 2, 3, 4, 31). The hemostatic-inflammatory response is immediate, with accumulation of thrombus and platelets and recruitment of neutrophils, lymphocytes, and monocyte-macrophages. Reepithelialization of the epidermis begins within hours of injury, and within 2–3 d granulation tissue formation commences, characterized by increased stromal cellularity and neovascularization. After this fibroproliferative process, extracellular matrix secretion and tissue remodeling occur, involving both activated fibroblasts (myofibroblasts) and macrophages. The complete process for normal first intention wound healing requires 2–3 wk. Thus, we determined changes in PAPP-A, IGF-I, and IGFBP-4 expression over this timeline (d 2, 8, and 14 postwounding). Furthermore, we assessed colocalization with myofibroblastic and endothelial cells using -SMA and with macrophages using CD68.

Figure 2 presents representative skin sections of PAPP-A staining 2, 8, and 14 d postwounding. Semiquantitative analyses of the immunohistochemistry results are summarized in Table 1. The most striking finding was the dynamic change in PAPP-A expression in the dermis. Thus, PAPP-A staining was detectable in the dermis on wound d 2, was uniformly strong on d 8, and was most intense on d 14. In general, staining was associated with increased number of cells and small blood vessels in the granulation tissue as well as with the more compact, amorphous mass of newly synthesized collagen. PAPP-A was expressed by cells with the spindle-shaped morphology typical of activated wound fibroblasts that were quite evident on d 8 (Fig. 3C). -SMA protein appeared de novo on d 8 after wounding in these myofibroblasts (Fig. 2; Fig. 3, B and D; and Table 2). In normal skin, -SMA was detectable in blood vessels, musculi arrectores pili, and myoepithelial cells of sweat glands (Table 2). PAPP-A staining was also prominent in activated wound macrophages, the numbers of which increased in wound tissue over time. These plump histiocyte-macrophages stain for -SMA as well as for the macrophage marker, CD68 (Fig. 4 and Table 2). PAPP-A staining of lymphoid cells was negative. The expression of PAPP-A in small blood vessels increased with further neovascularization, but the intensity of staining in individual endothelial cells showed no apparent change. There was some increase in PAPP-A staining intensity associated with reepithelialization of the epidermis. This is most apparent in the bridging projection or "tongue" of the healing epidermis in Fig. 2. There was no apparent change in PAPP-A expression in the sweat glands, sebaceous glands, or hair follicles in the periwound region at any time after injury (not shown).

View larger version (99K): [in this window] [in a new window]   FIG. 2. PAPP-A and -SMA immunostaining in human skin 2, 8, and 14 d post wounding. Magnification, x60. Solid arrows, epidermis; open arrows, dermis.

View larger version (133K): [in this window] [in a new window]   FIG. 4. Immunostaining of activated macrophages in granulation tissue for PAPP-A (A) and CD68 (B) on d 8 healing human skin. PAPP-A staining is associated with plump histocytic macrophages (indicated by arrows), a cell type that also stains positively for CD68. Magnification, x300.

View larger version (58K): [in this window] [in a new window]   FIG. 5. IGF-I immunostaining in human skin 2, 8, and 14 d post wounding. Magnification, x60. Arrows indicate the epidermis at the wound site on d 2.

View larger version (56K): [in this window] [in a new window]   FIG. 6. IGFBP-4 immunostaining in human skin 2, 8, and 14 d post wounding. Magnification, x60. Arrows indicate the epidermis at the wound site on d 2.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The appearance of PAPP-A in the granulation tissue by the second day after injury correlates with the proliferation and migration of fibroblasts and blood capillaries at the wound site (1, 2, 4). IGF-I and IGFBP-4 were also found in the granulation tissue at this time. Up-regulation of IGF-I mRNA and protein in wound tissue and detection in wound fluid has been documented in several systems (6, 7, 9, 10). IGFBP-4 is also present in wound fluid (37), but IGFBP-4 immunolocalization in skin has not been previously reported. The unusual nested areas of IGFBP-4 expression in epidermis observed in this study were not further investigated, but similar staining patterns have been reported for proteins with a rheostat role in cell growth control. Our results for IGFBP-4 are consistent with the in situ hybridization study performed by Batch et al. (20) in uninjured human skin. In their study the mRNA for IGFBP-4 localized to epidermis, sebaceous glands, sweat glands, and dermally derived fibrous root sheath. Thus, the presence of IGFBP-4 in the dermis and epidermis may play a regulatory role in IGF-I action throughout the skin.

The final stages of the fibroproliferative phase of wound healing are characterized by extracellular matrix production by fibroblasts accumulated in the cutaneous wound. After approximately 1 wk, there is abundant extracellular matrix, and the wound fibroblasts develop a phenotype with contractile properties. These myofibroblasts express -SMA and gain the capacity to generate contractile forces necessary for tissue remodeling (1, 2, 3, 4, 31). The colocalization of PAPP-A in granulation tissue with myofibroblasts on d 8 and 14 suggest a specific role for PAPP-A in tissue remodeling. These data coincide with in vitro studies showing abundant PAPP-A expression by vascular smooth muscle cells in culture (28). Interestingly, PAPP-A is expressed in granulation tissue on d 2 before the appearance of -SMA expression on d 8 in myofibroblasts. This suggests an additional cellular source of PAPP-A, such as macrophages, in the early phase of healing.

PAPP-A expression colocalized to activated macrophages in the periwound area. Macrophages are essential for effective and efficient wound healing through their ability to release factors that amplify the recruitment of inflammatory cells to the wound site, stimulate the formation of granulation tissue, and enhance cell migration. Macrophages have been shown to express IGF-I and IGFBP-4 in vitro (11, 12, 38), but there have been no studies addressing IGFBP-4 proteolysis or PAPP-A in these systems. It is of interest that PAPP-A also colocalized with vascular smooth muscle cells and macrophages in the inflammatory shoulder of vulnerable plaques in human coronary arteries (29). However, further studies are necessary to establish whether macrophages are a physiological source of PAPP-A.

Enhanced PAPP-A and IGF-I expression were localized to the advancing epithelial edge of the healing epidermis. This has been described previously in mice for IGF-I (7). Thus, focal and acute up-regulation of the IGF system may have a specific role in the rapid reepithelization of the epidermis to cover the wound.

These in vivo data are consistent with a model of PAPP-A amplification of local IGF-I bioactivity in wound healing based on the known information that human fibroblasts and vascular smooth muscle cells in culture produce IGFBP-4 and PAPP-A, and respond to IGF-I with increases in proliferation, migration, and extracellular matrix production (21, 25, 28, 35, 39). Thus, PAPP-A’s degradation of inhibitory IGFBP-4 would allow localized increases in IGF-I-mediated healing response. This is likely to involve paracrine regulation, because the cellular distribution of the three molecules is not completely coincidental in skin. Whether PAPP-A acts as an IGFBP-4 protease in this in vivo wound-healing situation could not be determined from these immunohistochemistry results, because the IGFBP-4 antibody detects both intact and fragmented IGFBP-4. Nonetheless, this study demonstrates specific localization and dynamic regulation of PAPP-A in human skin and suggests a potential function for PAPP-A as an IGFBP-4 protease in skin physiology and pathophysiology.

	Acknowledgments

 
We thank Ms. Kristen Shogren for her critical help with optimizing the immunohistochemistry.

	Footnotes

 
This work was supported in part by a fellowship grant (to B.-K.C.) from Diagnostic Systems Laboratories.

Abbreviations: IGFBP, IGF-binding protein; IgG, immunoglobulin G; PAPP-A, pregnancy-associated plasma protein A; -SMA, -smooth muscle actin.

Received February 6, 2003.

Accepted June 4, 2003.

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