Cyclopentyladenosine Improves Cell Proliferation, Wound Healing, and Hair Growth

Journal of Surgical Research 87, 14 –24 (1999) Article ID jsre.1999.5716, available online at http://www.idealibrary.com on

Cyclopentyladenosine Improves Cell Proliferation, Wound Healing, and Hair Growth 1 Leon L. Sun, Ph.D., M.D., 2 Linda L. Xu, M.D., Thor B. Nielsen, Ph.D., Peter Rhee, M.D., and David Burris, M.D. Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20852 Submitted for publication October 21, 1998

Conclusion. CPA stimulated proliferation of many cell types in vivo and in vitro and enhanced wound healing and hair growth. Therefore, CPA could be an interesting candidate for clinical application. © 1999 Academic Press Key Words: bromodeoxyuridine; angiogenesis; wound strength; hair follicle.

Background. N 6-Cyclopentyladenosine (CPA), a structural analog of adenosine, is a vasodilator with extensive pharmacological effects. However, little is known about the effect of CPA on wound healing and hair growth. Methods. Cellular responses to CPA were measured in vitro by tetrazolium dye reduction and in vivo by bromodeoxyuridine (BrdU) uptake. The effect of CPA on healing of incisional and excisional wounds on the dorsum of diabetic (db/db, n 5 94) and nondiabetic (db/1, n 5 20) mice and hair growth along the wound margin was evaluated with wound breaking strength, wound closure rate, and quantitative histology. Results. CPA stimulated proliferation of BALB/3T3 fibroblasts and human dermal microvascular endothelial cells in both quiescent and nonquiescent phases. Wounds treated with CPA at 10 mM showed a significant increase in the number of BrdU-labeled cells, including keratinocytes, fibroblasts, endothelial cells, and cells in sebaceous glands and the outer root sheath of hair follicles, compared with controls (P < 0.05). CPA application (5.1 mg/daily for 12 days) significantly increased the breaking strength of incisional wounds at day 24 postwound (P < 0.05). Excisional wound closure rate in the CPA-treated group (3.4 mg/ daily for 15 days) was accelerated starting at day 10 postwound compared with controls (P < 0.01). Tissue sections from CPA-treated wounds showed a sevenfold increase in hair follicle number, compared with controls (P < 0.01). Enhanced hair growth along the wound margin was revealed in CPA-treated groups.

INTRODUCTION

Tissue injury associated with ischemia results in release of mitogens that are sequestered within tissues or cells [1]. In many tissues, one consequence of cellular damage is release and degradation of ATP which is rapidly converted to adenosine. By binding to adenosine receptors and coupling to adenylate cyclase via G proteins, adenosine is involved in the control of many cellular events. For example, adenosine is a potent regulator of the inflammatory response, which is a component of wound healing [2]. Cyclopentyladenosine (CPA), a structural analogue of adenosine, is a highly selective adenosine A1 receptor agonist. By binding to the A1 receptors that are widely spread among almost all cell types, CPA has been shown to improve myocardial ischemia [3–7] and decrease cytosolic Ca 21 and coronary tension [8]. CPA also mediates the production of nitric oxide and the network of inflammatory cytokines such as IL-10 and TNF-a, which is important for tissue repair [9 –13]. However, little is known about the effect of CPA on wound healing. The extensive pharmacology of adenosine [14 –17] and the evidence of the therapeutic role of CPA in the processes of ischemia, vasorelaxation, and mediation of the release of growth factors led us to the hypothesis that exogenous application of CPA may play a role in the regulation of wound healing. We therefore tested the effect of CPA on cell proliferation in vitro, determined the dose–response relationship,

1 The project was funded by the Naval Medical Research and Development Command (61153N MR04120.001-1421 and G0190EP). The authors gratefully acknowledge Mr. Fleetwood A. Henry and HM3 Yusef Miller for excellent technical assistance. 2 To whom correspondence should be addressed at CPDR, Department of Surgery, Uniformed Services University of the Health Sciences, 1530 East Jefferson Street, Rockville, MD 20852. Fax: (240) 453-8912. E-mail: [email protected].

0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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SUN ET AL.: CPA IMPROVES HEALING AND HAIR GROWTH

TABLE 1 Animal Grouping Assay Cell proliferation

Breaking strength Wound closure a

Animals receiving CPA

Animals receiving PBS (control)

Strain a

Wound type

Days postwound for specimen harvesting

5 5 5 5 5 22 10

5 5 5 5 5 22 10

db/1 db/1 db/db db/db db/db db/db db/db

Incision Excision Incision Excision Incision Incision Excision

3 3 6 6 17 24 17

db/1, C57BL/KsJ db/1; and db/db, C57BL/KsJ db/db.

and then examined the effect of CPA on cellular responses, wound healing, and hair growth in diabetic and nondiabetic mice. MATERIALS AND METHODS

Cell Proliferation Assay in Vitro BALB/3T3 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT). Human dermal microvascular endothelial cells (HDMEC; Cell Applications, Inc., San Diego, CA) were maintained in basal medium for clonal growth and differentiation 131 (MCDB 131; Sigma Chemical Co., St. Louis, MO) supplemented with 10% FBS, 1 mg/ml hydrocortisone, and 10 ml/ml endothelial cell growth factor (Sigma). For the proliferation assay of cells in log growth (G1) or quiescent (G0) phase, the cells were harvested at 60 – 80% of confluence, seeded at a density of 5 3 10 3 cells/well into Falcon 96-well plates in DMEM (for BALB/3T3 cells) and in MCDB 131 (for HDMEC) supplemented with 10% FBS, and incubated either for 2 days to establish nonconfluent cells in log growth phase or for 6 days to establish confluent cells in quiescent phase. The cell monolayer was then rinsed with DMEM or MCDB 131 (without FBS), and the medium was changed to DMEM (for BALB/3T3) containing 20 mg/ml bovine serum albumin (Sigma), 5 mg/ml transferrin, and 2 mg/ml insulin (Gibco BRL) or to MCDB 131 medium (for HDMEC) supplemented with 1 mg/ml hydrocortisone and 10 ml/ml endothelial cell growth factor. At the same time, CPA (Sigma) was added at several concentrations. After 2 days of incubation with CPA, cell proliferation was measured by the reduction of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT; Sigma) as previously described [18].

Evaluation of CPA Effect in Vivo The experiments reported herein were conducted according to the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council, DHHS, Pub. No. (NIH)86-23 (1996), and were approved by the Institutional Animal Review Board. Genetically healing-impaired diabetic (db/db, n 5 94) and their normal littermate (db/1, n 5 20) mice (C57BL/KsJ, female, 8 weeks old, The Jackson Laboratory, Bar Harbor, ME) were divided into three groups (Table 1). Cell proliferation assay. In vivo incorporation of 5-bromo-29deoxyuridine (BrdU; Sigma), an analog of thymidine, followed by immunostaining of tissue sections was used to identify cells in S-phase (DNA synthesis phase) and to assess cell proliferation in

tissues [19]. Diabetic mice and their normal littermates were divided into four subgroups (Table 1). In the incisional wound groups, a single full-thickness incision 2 cm long was made on the back of shaved mice and closed by 3-O sutures [18, 20]. Each incisional wound received 15 ml/dose (5.1 mg/dose) (10 ml injected into the wound base and 5 ml topically applied to the wound margin) of 10 mM CPA in PBS once daily. In the excisional wound groups, a single full-thickness 6-mm diameter excisional wound was made on the back with a sterile biopsy punch (Acuderm, Inc., Ft. Lauderdale, FL). Each wound received 10 ml of 10 mM (3.4 mg/dose) CPA in PBS applied topically once daily. The corresponding controls received PBS and were processed identically. To prevent disturbance of wounds by licking, animals were individually housed after creation of wounds. The wound tissue was harvested at the indicated days, with each animal receiving a single injection (ip) of BrdU at a dosage of 50 mg/kg body weight 2 h before death.

TABLE 2 CPA Stimulated in Vitro Cell Proliferation in Quiescent and Nonquiescent Phases BALB/3T3 cells CPA (mM) 0.1 0.5 1 2 5 10 20 40

G0 phase

G1 phase

1.04 6 0.04 1.06 6 0.02 1.05 6 0.04 1.07 6 0.06 1.08 6 0.06 1.09 6 0.04 1.09 6 0.07 1.12 6 0.07 1.13 6 0.09* 1.22 6 0.05* 1.13 6 0* 1.06 6 0.03 1.16 6 0.03* 1.00 6 0.02 1.12 6 0 Not determined

HDMEC G0 phase

G1 phase

1.12 6 0.03* 1.08 6 0.04* 1.08 6 0.01* 1.06 6 0.01 1.07 6 0.03 1.04 6 0.05 1.04 6 0.02 0.98 6 0.08

1.01 6 0.04 1.12 6 0.03* 1.14 6 0* 1.18 6 0.02* 1.12 6 0.03* 0.90 6 0.04 0.92 6 0.03 0.87 6 0.01

Note. BALB/3T3 fibroblasts and human dermal microvascular endothelial cells (HDMEC) were cultured for 2 days to obtain nonconfluent monolayers (G1 phase or log growth phase) or for 6 days to obtain confluent monolayers (G0 phase or quiescent phase), then incubated with CPA for 2 days. Cell proliferation was measured by the reduction of MTT. The data were means (6SD) of six wells of two separate experiments and presented as a ratio of the OD values (CPA treated:PBS control). The OD values of PBS control were 0.48 6 0.05 in G0 phase group and 0.36 6 0.04 in G1 phase group for the BALB/3T3 cells and 0.50 6 0.03 in G0 phase group and 0.42 6 0.03 in G1 phase group for the HDMEC. The cell numbers of PBS control were 52.2 6 2.8 (310 3) in G0 phase group and 20.0 6 0.9 in G1 phase group for the BALB/3T3 cells and 42.9 6 2.1 in G0 phase group and 21.6 6 0.4 in G1 phase group for the HDMEC. * P , 0.05, compared with corresponding control.

FIG. 1. Dynamic distribution of proliferating wound cells in response to CPA. (A) BrdU-labeled cells (arrowheads) located mainly in the basal membrane in a CPA-treated incisional wound at day 2 postwound in a nondiabetic mouse (db/1). (B) BrdU-labeled cells located in the basal membrane, as well as in the dermis and sebaceous glands in a CPA-treated db/1 incision wound at day 3 postwound. (C) BrdU-labeled cells located mainly at the front of the wound margin in a CPA-treated db/1 incision wound at day 4 postwound. (D) BrdU-labeled cells distributed in the outer root sheath of a hair follicle (arrowheads) in a CPA-treated wound in a db/db mouse at day 7 postwound. The a, b, c, d, and e denote the epidermis, dermis, sebaceous gland, wound margin, and hair follicle, respectively. The bar represents 100 mm.

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FIG. 2. Enhancement of endothelial cell proliferation and angiogenesis. (A) BrdU-labeled S-phase endothelial cells around the wall of neocapillaries (arrowheads) in an excisional wound at day 7 postwound in a diabetic mouse (db/db) and (B) angiogenesis (red staining of Factor VIII-related antigen, arrowheads) in a db/db excisional wound at day 7 postwound. The bar represents 50 mm in A and 200 mm in B. Breaking strength measurement in incisional wounds. A fullthickness incisional wound as described above was made in db/db mice. The wound received 15 ml/dose (10 ml injected at the base of the incision and 5 ml topical application on the incision margin) of either 10 mM CPA in PBS or PBS vehicle (as control) once daily for 12 days. Five wounds each from the CPA and PBS groups were excised at day 17 postwound, and the remaining wounds were harvested at day 24 (Table 1). The wound skin was divided into two parts: one was fixed in 10% formalin and the other was cut into 8-mm-wide strips using a template. Breaking strength was measured immediately with the 8-mm wound strips using a custom-made tensiometer. Tension was applied at a constant rate of 1 cm/min using a 1.0 kg force transducer. Breaking strength, the point of maximal stress before wound

separation, was recorded and analyzed on a computer equipped with custom-made software [20]. Wound closure measurement in excisional wounds. A single fullthickness excisional wound as described above was made on the backs of 20 female diabetic mice (Table 1). The wounds were treated with either 10 ml/dose of 10 mM CPA in PBS or the PBS vehicle (as control) once daily for 15 consecutive days postwound. Scabs were gently removed on days 4, 6, 8, 10, 12, 14, and 16 postwound to accurately visualize the wound margin and to facilitate access of the test agent to the wound. The wound area was recorded with a color video camera and a specially designed mouse chamber at 1 h postwound (day 0) and alternate days thereafter. The size of the wound

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was amplified to about 100 –200 times the original and analyzed by using an image analysis system (BioScan, Inc., Edmonds, WA) [20].

Tissue Processing and Quantitative Assessment To visualize BrdU incorporation and angiogenesis, immunostaining was performed on 5-mm-thick tissue sections from the wound at day 2–7 postwound with monoclonal antibodies against BrdU or factor VIII-related antigen (Zymed Laboratories, Inc., South San Francisco, CA) by following the manufacturer’s procedures. Skin samples harvested from the breaking strength measurement group were embedded in paraffin and stained with hematoxylin and eosin. Quantitative evaluation of the CPA effect on wound healing was performed on all sections with a Nikon Optiphot microscope interfaced via a color camera to the image analysis system, by measurement of BrdU-labeled cells, cellular density, thickness of dermal and adipose layer, and hair follicles in the wound site. The data are the means of two fields/section of two sections/incision wound from all animals in each group.

Data Analysis Data are presented as means 6 standard deviation. Statistical analysis of the data was performed by using the one-way ANOVA test, except as noted.

RESULTS

Enhanced Cellular Responses to CPA in Vitro Proliferation of BALB/3T3 fibroblasts and human dermal microvascular endothelial cells was analyzed to evaluate cellular responses of different wound cell types to CPA and to choose an effective dose range of CPA for the in vivo studies. For cells in log growth phase (nonquiescent), CPA was modestly, but significantly stimulatory (Table 2), with a peak response at 5 mM (for fibroblasts) and 2 mM (for endothelial cells). For fibroblasts and endothelial cells in quiescent phase, CPA was stimulatory at low concentrations (Table 2). Enhanced Cellular Responses to CPA in Vivo Healing was faster in nondiabetic db/1 mice than in diabetic mice. In the db/1 mice, therefore, the distribution of the BrdU-labeled proliferative cells identified by the purple nuclei changed rapidly with the length of time after wounding (Figs. 1A–1C). At day 2, proliferating cells were mainly keratinocytes located in the basal membrane of the epidermis (Fig. 1A). The enhanced proliferation of keratinocytes occurred over a span of about 200 cell diameters in the tissue adjacent to the wound edge. At day 3 the proliferating cells occurred in the dermis, sebaceous glands, and hair follicles, while the number of proliferating cells in the basal membrane was decreased (Fig. 1B). At day 4 the proliferating cells were located mainly at the wound margin (Fig. 1C). The migration of proliferating cells toward the wound revealed the dynamic process of tissue repair. A wide variety of cells including keratinocytes, fibroblasts, and cells around sebaceous glands

TABLE 3 CPA Stimulated in Vivo Cell Proliferation in Normal and Genetically Healing-Impaired Mice Diabetic mice (db/db) Incision CPA 76.6 6 41.8* PBS 48.2 6 19.1

Wild-type mice (db/1)

Excision

Incision

Excision

139.6 6 40.8* 68.9 6 65.7

104.9 6 35.3* 60.2 6 34.2

96.1 6 59.3* 52.6 6 27.6

Note. The wounds were treated either with CPA or PBS daily and harvested at day 3 for db/1 and 6 for db/db mice. The BrdU-labeled cells were counted with an image analysis system under a 103 objective lens (0.245 mm 2/field). * P , 0.05, compared with corresponding PBS controls.

appeared to have incorporated BrdU in response to CPA. In addition, the cells around the outer root sheath of hair follicles actively responded to CPA stimulation as demonstrated by BrdU incorporation (Fig. 1D). An effect of CPA on endothelial cells was also observed. In CPA-treated excisional wounds in db/db mice, BrdU was incorporated into endothelial cells of neocapillaries (Fig. 2A). The intense red staining of Factor VIII-related antigen (Fig. 2B) supported the observation that CPA stimulated endothelial cell proliferation and angiogenesis. Quantitative evaluation of BrdU-labeled cells showed that CPA treatment significantly enhanced cell proliferation both in incisional and in excisional wounds of db/1 and db/db mice (P , 0.05, Table 3). By these measures, CPA was a potent mitogen in wounds. Improved Incisional Wound Healing CPA enhanced BrdU incorporation and formation of granulation tissue in the incisional wounds of diabetic mice (Figs. 3A and 3B). Control wounds receiving PBS, however, had few BrdU 1 cells and an appearance of poor healing with little granulation tissue (Fig. 3C). CPA treatment resulted in a significant improvement in the breaking strength of the wounds measured at day 24, compared with the PBS control (P , 0.05, Table 4). The breaking strengths of wounds treated with CPA and PBS were not significantly different at 17 days postwound, although the breaking strength in the CPA-treated group seemed higher than in the PBStreated group (Table 4). Quantitative assessment of the histology of the CPA-treated wounds 24 days postwound revealed several statistically significant changes (Table 5). The cellular density was increased by over 30% (P , 0.01). The thickness of the dermis was about one-quarter that of the control thickness (P , 0.01). Of note, the number of hair follicles in the wound area increased by over sevenfold (P , 0.01). Thus, several measures of the quality of healing were improved by CPA treatment.

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Accelerated Excisional Wound Closure

Role of CPA in Wound Healing

The full-thickness lesions in diabetic mice may simulate deep dermal decubitus and diabetic ulcers in humans. Such wounds frequently penetrate through the dermis and are often debrided to remove necrotic tissue prior to treatment, thereby generating a deep wound bed similar to that produced by punch biopsy in rodents. Measurement of the wound areas demonstrated that CPA treatment accelerated the wound closure rate at day 10 and thereafter, when normalized to controls and compared to the initial areas (P , 0.01, Table 6). Comparison of the absolute areas for CPA vs PBS, rather than the ratios, also demonstrated a significant change, e.g., 6.5 6 2.9 mm 2 for CPA vs 12.1 6 4.2 mm 2 for PBS at day 10 (P , 0.01).

Adenosine is thought to exert its effects through specific exterior cell surface receptors that can be subdivided into three classes: A1 and A3 receptors that inhibit adenylate cyclase and A2 receptors that stimulate adenylate cyclase. These receptors have been classified by their differing affinities for adenosine and its structural analogues. The A1 adenosine receptors exhibit an agonist potency order of CPA . R-N 6phenylisopropyladenosine (R-PIA) . adenosine . 59N-ethylcarboxamidoadenosine (NECA) . 59-N-(cyclopropyl)carboxamindoadenosine (NCPCA), whereas for A2 receptors the order is reversed: NCPAC . NECA . adenosine . R-PIA . CPA [21–23]. Wound healing is a complex localized biological process involving proliferation of many cell types in the wound that may be responsive to CPA and contribute to the enhancement of healing. In addition, cells in the wound site may be randomly distributed in log phase (G1) or quiescent phase (G0). Therefore, we characterized the effect of CPA on examples of two major types of wound cells and their different parts of the cell growth cycle. Results of the dose–response assay in vitro indicated that fibroblasts and endothelial cells both in log phase and in quiescent phase responded to the CPA stimulus. Other in vitro experiments support the observation that adenosine is a mitogen for endothelial cells [24 –25]. The response of fibroblasts to adenosine A1 receptor agonists such as CPA is not described. However, it has been demonstrated that through the adenosine A2 receptor, adenosine inhibits synthesis of collagen and protein and fibroblast proliferation [26, 27]. The complexity of the responses to adenosine may derive, in part, from the complex roles of adenosine in activation and inhibition of cellular second-messenger systems [14 –17]. The mechanism of the stimulation may be by suppression of cyclic AMP levels in the cells. Agents that elevate cyclic AMP levels, including IBMX (a phosphodiesterase inhibitor) and NCPCA (A2 receptor anonist), lead to suppression of porcine cerebral microvascular endothelial cell proliferation. Similarly, forskolin, which stimulates adenylate cyclase and leads to elevated levels of cAMP, inhibited growth of brain microvessel endothelial cells [28]. Cell proliferation in vivo is more complex than in vitro and may have contributions from any of several factors, including addition of a mitogen, the presence of

Promoted Hair Growth along the Wound Margin In most CPA-treated diabetic mice, new hair growth was visible in the immediate area of the wounds at day 16 and thereafter, while the corresponding PBS controls showed significantly less frequent hair growth (Fig. 4A). This phenomenon was also observed in the normal littermates (db/1) of the diabetic mice (Fig. 4B). Some hair growth was not infrequent in wound sites in adult wild-type strains of mice such as db/1 mice, but less common in adult diabetic mice. Histology revealed a high density of large proliferative hair follicles in the CPA-treated wound of db/db mice (Fig. 4C), but not in the PBS-treated wound (Fig. 4D). The incidence of CPA induction of hair growth was quantified and found to be significant (P , 0.0001, Table 7), compared with PBS controls, consistent with the data of cell proliferation around hair follicles (Fig. 1D) and the increase in the number of hair follicles (Table 5) observed in CPA-treated wounds. DISCUSSION

Two important effects of CPA are demonstrated: (1) enhancement of wound healing and improvement of the quality of healing by the criteria of quantitative histology and immunohistochemistry, wound strength, and wound closure rate and (2) stimulation of hair growth by quantitation of hair follicle number. The mitotic effects of CPA both in vitro and in vivo may suggest a mechanism of action.

FIG. 3. Improvement of in vivo cell proliferation and incisional wound healing. (A) Many BrdU-labeled cells (arrowheads) distributed in the epidermis, dermis, subcutaneous layer, and wound site in a CPA-treated incisional wound harvested at day 7 postwound in a diabetic mouse (db/db). The frame indicates the size (0.245 mm 2) of a field for counting BrdU cells. (B) High magnification of A, showing the BrdU-labeled cells (arrowheads) in the epidermis and the wound site, complete epithelialization, an increase in granulation tissue in wound site, and a better appearance of healing. (C) Little granulation tissue and the appearance of poor healing in a PBS-treated incisional wound (control of A and B). The a, b, c, and d denote the epidermis, dermis, adipose layer, and wound site, respectively. The bar represents 400 mm in A and C and 200 mm in B.

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FIG. 4. Stimulation of hair growth. (A) Hair growth (arrows) along the margin of a CPA-treated (10 ml of 10 mM once a day for 15 days) excisional wound in a diabetic mouse (db/db) at day 44 postwound. (B) Hair growth (arrows) along the margin of a CPA-treated (15 ml of 10 mM once a day for 12 days) incisional wound in a nondiabetic mouse (db/1) at day 19 postwound. (C) Tissue section showing a high density of hair follicles (arrowheads) and normalized complement of fat cells in the subcutaneous layer near the wound site in a CPA-treated incisional wound in a db/db mouse at day 24 postwound. (D) Few hair follicles, but a large amount of granulation tissue in a PBS-treated incisional wound (control of C). The bar represents 200 mm.

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TABLE 4

TABLE 5

CPA Enhanced Breaking Strength of Incisional Wounds

CPA Improved Healing Quality and Hair Growth

Breaking strength (g) at 17 days postwound

Breaking strength (g) at 24 days postwound

261 6 11 233 6 77

497 6 108* 363 6 82

Note. The full-thickness incisional wounds on db/db mice were treated with either CPA (15 ml of 10 mM) or PBS daily for 12 days postwound and harvested at the day indicated. The means (6SD) of the breaking strength were from up to two strips/wound for all animals in each group. * P , 0.05, compared with the PBS control.

costimulators, and improvement of impaired blood perfusion. The current report clearly demonstrates that CPA stimulated proliferation of keratinocytes, fibroblasts, and endothelial cells in vivo. In addition to a role as mitogen, CPA has potent vasodilatory effects [8, 29] that may contribute to the effects on cell proliferation in vivo and on wound healing, perhaps by modulation of local blood perfusion of the wound tissue. The changes of cellular responses may explain the enhancement of biomechanical features and the acceleration of wound closure in the CPA-treated wounds. The fibroblast proliferation in wound tissue may be induced by CPA acting directly as a mitogen or indirectly through an interaction between endothelial cells and fibroblasts, the CPA-mediated release of regulatory factors from endothelial cells, or improvement of vascularization. For example, a number of putative angiogenic factors including small molecules (e.g., adenosine) have been shown to upregulate the expression of vascular permeability factor/vascular endothelial growth factor [30]. The acceleration of wound closure induced by CPA treatment may also result from an acceleration of epithelialization in the wound site by proliferation of keratinocytes in the wound edge instead of wound contraction. Similarly, the keratinocyte proliferation may be an indirect action of CPA on blood flow, a cell type with adenosine receptors, or a cytokine cascade involving the upregulation of kerotinocyte growth factor. Breaking strength in CPA-treated wounds increased, but did not reach a significant level of improvement in the wounds harvested at 17 days postwound, probably because 17 days was insufficient for collagen production and remodeling in the wound site. Stimulating Effect of CPA on Hair Growth Even though there is little precedent with respect to the relationship between CPA and hair growth, the hair growth induced by CPA along the wound margin suggested a novel mechanism that may be different

Cellular density (0.1 mm 2)

Dermis thickness (mm)

Hair follicles (per 1000 mm)

564 6 31* 362 6 82

360 6 100* 1380 6 260

31 6 13* 4.1 6 3.7

CPA PBS

Note. The incision wounds in db/db mice were treated with either CPA (15 ml of 10 mM) or PBS daily for 12 days and harvested at day 24 postwound. By using an image analysis system, the hair follicles in the wound margin 1000 mm long were examined under a 43 objective lens, the dermis thickness was measured under a 203 objective lens, and the cellular density was measured under a 403 objective lens. * P , 0.01, compared with corresponding controls.

from, or a component of, those that explain the effect on wound healing. One of the mechanisms of the hair growth induced by CPA may be the vasorelaxant effect of CPA [8, 29]. Minoxidil, the active ingredient in the commercial product Rogaine available for hair growth, is also a vasodilator [31–33]. The marked angiogenesis induced by CPA may also be related to the induced hair growth. According to current understanding, the cell proliferation in dermal papilla has a crucial role in hair follicle development and regeneration during the hair cycle, and epidermal cells have a “conditioning action” for hair follicle formation [34 –36]. The increase in the number of the proliferating keratinocytes and cells located in the sebaceous glands and outer root sheath of hair follicles is consistent with a direct action of CPA as a mitogen for these cells in the dermis and epidermis and with an indirect effect of CPA as a vasodilator and angiogenesis enhancer. The hair follicle proliferation in CPA-treated wounds TABLE 6 CPA Accelerated Closure Rate of Excisional Wounds Day after wounding

CPA (% control)

PBS control (mm 2)

0 2 4 6 8 10 12 14 16

100 6 17 112 6 7 99 6 11 87 6 9 88 6 17 54 6 24* 44 6 17* 15 6 17* 7 6 15*

32.8 6 4.9 33.4 6 5.6 31.3 6 5.0 24.5 6 3.8 17.2 6 2.1 12.1 6 4.2 7.5 6 3.3 6.4 6 2.7 4.4 6 2.4

Note. The time courses of wound areas were examined in db/db mouse excision wounds treated with either CPA (10 ml of 10 mM) or PBS (control of CPA group) daily. There were 10 mice in each group. The data represent two separate experiments. The means (6SD) of the wound areas were expressed as percentages of the areas of control wounds. * P , 0.01, compared with the value at day 0 by the one-way ANOVA test.

SUN ET AL.: CPA IMPROVES HEALING AND HAIR GROWTH

TABLE 7

9.

CPA Stimulated Hair Growth along Wound Margin Incisional wound

CPA PBS

Excisional wound

db/1 mice

db/db mice

db/db mice

P value a

4/5 2/5

16/22 5/18

8/10 1/10

,0.0001

Note. Incisional or excisional wounds were treated with either CPA or PBS. The data were expressed as the number of wounds with hair growth of the total number of wounds in each group. a Compared to all wounds with hair growth between the CPA- and the PBS-treated groups with x 2 test.

10.

11.

12.

13.

may not only be a potential bonus to the mending of injuries or surgical incisions on the scalp, eyebrows, upper lips, and so on, but may also contribute to wound remodeling, wound tissue normalization, and decrease in the amount of scar tissue as seen in this study. These results suggest that CPA may improve the quality of healing. In conclusion, CPA stimulated proliferation of many cell types in vitro and in vivo, enhanced healing of incisional and excisional wounds, and improved hair follicle proliferation and the quality of healing in both normal and genetically healing-impaired mice. Therefore, CPA could be an interesting candidate for clinical application.

14.

15.

16.

17.

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