Topical Pretreatment of Diabetic Rats With All-trans Retinoic Acid Improves Healing of Subsequently Induced Abrasion Wounds
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糖尿病学杂志 2005年第3期
1 Department of Pathology, University of Michigan, Ann Arbor, Michigan
2 Department of Internal Medicine, the University of Michigan and the Ann Arbor Veterans Administration Hospital, Ann Arbor, Michigan
ABSTRACT
In the current study, rats were made diabetic with streptozotocin (STZ) and maintained for 8 weeks, during which time they were treated topically on alternative days with a solution of 0.1% all-trans retinoic acid in a vehicle of 70:30% ethanol/propylene glycol. STZ-induced diabetic rats treated with vehicle served as controls. Additional nondiabetic rats were treated with all-trans retinoic acid or vehicle in parallel. At the end of the 8-week period, rats from all four treatment groups were subjected to abrasion wound formation. Wounds healed more rapidly in vehicle-treated nondiabetic skin than in vehicle-treated diabetic skin (96% of the wound surface area closed in nondiabetic rats within 6 days vs. 41% closed in diabetic rats). Wounds in all-trans retinoic acid-treated diabetic skin healed more rapidly than wounds in vehicle-treated diabetic skin (85% of the wound surface area closed in all-trans retinoic acid-treated diabetic rats vs. 41% closed in vehicle-treated diabetic rats). At the histological level, recently healed skin from vehicle-treated diabetic rats was shown to contain a thin, wispy provisional matrix in which many of the embedded cells were rounded and some were pycnotic. In contrast, a much denser provisional matrix with large numbers of embedded spindle-shaped cells was observed in healed wounds from diabetic skin that had been pretreated with all-trans retinoic acid. The all-trans retinoic acid-treated diabetic skin was histologically similar to vehicle-treated (or all-trans retinoic acid-treated) skin from nondiabetic animals. In light of these findings, we suggest that prophylactic use of retinoid-containing preparations might be useful in preventing the development of nonhealing skin ulcers resultant from minor traumas in at-risk skin.
Diabetes is responsible for delayed or impaired wound healing, leading in many instances to chronic ulcer formation. Diabetic ulcers of the lower limbs and feet, in particular, are associated with high morbidity and often lead to amputation (1eC3). Peripheral neuropathy and peripheral vascular disease are thought to be underlying factors in diabetic wound formation (2eC6), but dermal atrophy is likely to be a contributing factor in many cases (6,7). Reduced fibroblast growth, increased expression of matrix-degrading matrix metalloproteinases (MMPs), and decreased matrix synthesis are consequences of chronic vascular disease in diabetic skin as well as in other situations where peripheral vascular disease is present (7eC15). Atrophic skin is less resistant than healthy skin to wound formation. In addition, scanty extracellular matrix production during the proliferative phase of wound repair undoubtedly contributes to poor healing (16,17).
Past studies have shown that all-trans retinoic acid has the capacity to repair photodamaged skin (18eC19). The repair response includes both increased procollagen production and decreased elaboration of tissue-destructive MMPs. In addition, there is epidermal thickening caused by retinoid-induced keratinocyte proliferation. Chronologically aged skin demonstrates the same responses to all-trans retinoic acid (20). Our own recent studies have demonstrated that diabetic skin, including skin from the lower legs of individuals at risk for ulcer formation, also responds to retinoid treatment with increased thickening, increased collagen production, and most dramatically, decreased levels of collagen-degrading MMPs (including interstitial collagenase [MMP-1] and gelatinase B [MMP-9]) (21,22). Reduction in MMP levels reflects decreased enzyme production and, especially, a reduction in enzyme activation, subsequent to an increase in TIMP-1 (tissue inhibitor of metalloproteinases-1) production in the retinoid-treated skin (22). Based on these data, we have hypothesized that prophylactic treatment of skin with all-trans retinoic acid might produce changes in the skin that would enhance wound healing and, thereby, increase resistance to chronic ulcer formation. The current studies describe efforts to test this hypothesis in an experimental model of diabetes in rats.
RESEARCH DESIGN AND METHODS
Male nude rats with a start weight of 200eC250 g were purchased from Charles River (Wilmington, MA) and acclimated for 1 week on arrival in the laboratory. Animals were randomized to control or treatment groups. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 50 mg/kg in 0.2 ml of 10 mmol/l citrate buffer, pH 5.5; Sigma, St. Louis, MO). After 48 h, blood glucose levels were assessed, and rats were included in the study if glucose concentrations were >250 mg/dl in heparinized tail vein blood (measured by glucometer). The university committee on use and care of animals approved all of the procedures used in this study.
Topical treatment with all-trans retinoic acid or vehicle.
Diabetic rats and an equal number of nondiabetic controls were divided into treatment groups. Starting 3 days after STZ administration, one group (diabetic and nondiabetic) received 100 e蘬 of a 0.1% solution of all-trans retinoic acid in 70:30% ethanol/propylene glycol, applied topically to the dorsal flank skin on both sides. Treatment was every other day for 8 weeks. A second group of diabetic and nondiabetic rats was treated with vehicle alone over the same period. All treated and control rats had ad libitum access to water and suitable rat diet. STZ-treated rats were given daily injections of a small dose of protamine zinc insulin (0.5eC3.0 units per day) as required to maintain blood glucose levels between 350 and 500 mg/dl. At the end of the 8-week treatment, four rats from each of the four groups were killed. Skin samples from the retinoid-treated or vehicle-treated sites were fixed in 10% buffered formalin and used for histology. The remainder of the rats were included in wound-healing studies.
Superficial wound formation protocol.
Rats were anesthetized with an intraperitoneal injection of ketamine/xylazine (100 mg/kg ketamine and 10 mg/kg xylazine), and the paravertebral skin was cleansed with 70% alcohol. Abrasion-type superficial wounds were made on the paravertebral dorsal skin by application of 100% acetone to the skin followed by rubbing with a coarse emery board. A circular wound area with approximate dimensions of 6 x 6 cm was made. The depth of the wound was made just to the point where oozing of fluid into the abraded tissue could be seen. The superficial wounds were designed to mimic the type of wound typically seen in human skin after minor scrapes.
Wounds were photographed using a digital camera on day 0 and on days 2, 4, and 6. Wound area was calculated as a multiple of x- and y-axes of the wound. At the time of wound closure, animals were killed by cervical dislocation. Duplicate pieces of dorsal flank skin from three different sites (i.e., wound center, wound edge, and beyond the original wound margin) were taken from each animal. One of the duplicate biopsies was immediately fixed in 10% buffered formalin and used for histology. The second piece was placed in organ culture (see below).
Organ culture.
Biopsies for organ culture were cut into pieces 2 mm on a side and incubated in wells of a 24-well dish. Five to six tissue pieces were placed in each well along with 1 ml of culture medium, consisting of Dulbecco’s modified minimal essential medium containing 200 e/ml BSA. Cultures were incubated at 37°C in an atmosphere of 5% CO2 and 95% air. Organ culture-conditioned medium collected on day 3 was used for the assessment of MMP and fibronectin levels as described below. Organ culture of rat skin has been described previously (21).
Substrate-embedded enzymography.
Substrate-embedded enzymography (zymography) (23) was used to identify and characterize MMPs. Briefly, SDS-PAGE gels were prepared from 30:1 acrylamide/bis with the incorporation of gelatin (1 mg/ml) before casting. The gels were routinely 7.5% acrylamide. Samples of organ culture fluid and molecular weight standards were electrophoresced at a constant voltage of 150 volts under nonreducing conditions. After electrophoresis, gels were removed and washed twice for 15 min in 50 mmol/l Tris buffer containing 1 mmol/l Ca2+ and 0.5 mmol/l Zn2+ with 2.5% Triton X-100. The gels were then incubated overnight in Tris buffer with 1% Triton X-100 and stained with Brilliant Blue R concentrate the following day. After destaining, zones of gelatin hydrolysis were detected as clear areas against a dark background. Zymographic images were digitized. Negative images were created and quantified by scanning densitometry. Using the digitized images, quantitative values for latent and active MMP-2 (72-kDa gelatinase A) and MMP-9 were obtained.
Western blot analysis.
Western blotting was performed as described previously (21). Briefly, a mouse polyclonal antibody to rat fibronectin was obtained from Chemicon (Temicula, CA). Equivalent amounts of organ culture-conditioned medium were resolved by SDS-PAGE (7.5%) and transferred to a nitrocellulose membrane using a Bio-Rad minitransfer blotting apparatus. Membranes were then blocked in Ca2+- and Mg2+-free Tris-buffered saline (TBS) containing 5% blotto. Membranes were then treated with the primary antibody (1:200 dilution) in TBS containing 0.5% blotto (blotting-grade nonfat dry milk; Bio-Rad, Hercules, CA) and 0.1% Tween for 1 h. After several rinses in TBS with 0.1% Tween, the membrane-bound antibody was reacted with horseradish peroxidase-conjugated rabbit anti-mouse antibody at 1:2000 dilution for 1 h. Protein-antibody complexes were detected by enhanced chemiluminescence (Cell Signaling, Beverly, MA) and visualized on light-sensitive autoradiographic film (Amersham Biotechnology, London). Images were digitized and quantified as described above with zymograms.
RESULTS
Plasma glucose values and body weights of nondiabetic and diabetic rats.
Initially, 80 hairless rats were included in the study. Half of the rats were made diabetic by giving STZ as described in the RESEARCH DESIGN AND METHODS section. The diabetic and nondiabetic rats were divided into two groups each. One set of diabetic rats was treated with 0.1% all-trans retinoic acid on the paravertebral skin, and the second set of diabetic rats was treated with vehicle only. The two groups of nondiabetic rats received the same treatment as the diabetic rats. The mean body weights and blood glucose values for each group was measured on day 1 of treatment with all-trans retinoic acid and vehicle, on day 56 of treatment (immediately after the final treatment and before wounding), and immediately before they were killed (6eC8 days later). The data are shown in Table 1.
Histological features of skin from vehicle-treated and all-trans retinoic acid-treated nondiabetic and diabetic rats.
Rats from each of the four groups were observed for changes in the physical appearance of the skin at weekly intervals throughout the study. The major difference between vehicle-treated nondiabetic and diabetic rats was that in the diabetic rats, the skin became progressively paler and more translucent over the 8-week observation period. At the time they were killed, the skin of vehicle-treated diabetic rats had a "papery thin feel." No major change in the skin from the nondiabetic rats was observed over the same period. The changes observed in the skin of vehicle-treated diabetic rats were not observed in companion rats treated with all-trans retinoic acid. Rather, the skin of all-trans retinoic acid-treated rats remained similar in appearance to that of nondiabetic rats throughout the observation period. Consistent with what has been reported (24eC26), the skin of all-trans retinoic acid-treated hairless rats (both nondiabetic and diabetic) demonstrated a degree of irritation, consisting of redness, dryness, and flaking.
Figure 1 demonstrates histological features of skin from each of the four groups of rats at 8 weeks posttreatment. Comparing vehicle-treated nondiabetic and diabetic rats, differences between the two groups included a thinner epidermis and more pycnosis in the interstitial cell population of the diabetic rats. After retinoid treatment, the epidermis of both groups was significantly thickened, and the interstitial cells of the dermis were characterized by the presence of plump, oblong, light-staining nuclei (characteristic of actively metabolizing cells). Table 2 presents epidermal thickness measurements in the four groups of rats.
Wound formation and healing.
At the end of the treatment period, circular abrasion wounds with a diameter of 35 cm2 were created in the paravertebral skin at the all-trans retinoic acid- and vehicle-treated sites. Wound healing was followed and time to wound closure measured as described above. As seen in Fig. 2, the mean wound surface area on day 0 of wounding in the vehicle-treated diabetic rats was 34.8 ± 3.4 cm2 as compared with the mean wound surface area of 37.3 ± 4.9 cm2 in vehicle-treated nondiabetic rats. By day 6 postwounding, the mean wound surface area in vehicle-treated diabetic rats was reduced to 13.7 ± 2.0 cm2, whereas the mean wound surface area in the vehicle-treated nondiabetic rats was reduced to 1.3 ± 1.4 cm2.
In the all-trans retinoic acid-treated group of diabetic rats, the original mean wound area was 34.6 ± 4.5 cm2 as compared with the original mean wound surface area of 34.8 ± 3.4 cm2 in vehicle-treated diabetic rats. By day 6, the wound surface area of the all-trans retinoic acid-treated rats was 5.1 ± 2.8 cm2 as compared with 13.7 ± 2.0 cm2 in the vehicle-treated diabetic rats. Although the data in Fig. 2 shows wound area through day 6, in some of the vehicle-treated diabetic rats, wounds had not completely healed even by day 8 (not shown). In the nondiabetic rats treated with all-trans retinoic acid, the original wound surface area was 35.4 ± 3.4 cm2. By day 6, wounds in all of the all-trans retinoic acid-treated rats had completely healed. However, because even in vehicle-treated nondiabetic rats, wounds were almost completely healed by day 6 (wound surface area of 37.3 ± 4.9 cm2 on day 0 reduced to 1.3 ± 1.4 cm2 by day 6), differences between all-trans retinoic acid- and vehicle-treated nondiabetic rats were not statistically significant.
Figure 3 demonstrates gross and histological features of vehicle-treated and all-trans retinoic acid-treated diabetic skin. Grossly, there was no visible difference in the appearance of the wounds between the two groups while they were healing, although the still-scabbed-over wound on the vehicle-treated rat is apparent at day 6, whereas the wound of the all-trans retinoic acid-treated rat has healed completely. Histologically, the appearance of the wound site in both all-trans retinoic acid-treated and vehicle-treated rats was distinguishable from control sites (beyond the wound margin) by the presence of abundant provisional matrix beneath the epidermis (lightly stained extracellular matrix in the hematoxylin and eosin-stained sections). In general, the matrix was less organized in the vehicle-treated skin than in all-trans retinoic acid-treated skin. In addition, there were few spindle-shaped or elongated cells in the provisional matrix of the vehicle-treated skin, and many of the cells had a shrunken, rounded, pycnotic appearance. In contrast, in the all-trans retinoic acid-treated skin, the majority of cells appeared to be spindle shaped (consistent with the appearance of healthy fibroblasts). Although these differences were readily apparent in many samples, there was significant variability within each group, and we were unable to accurately quantify the differences between the two groups. Skin at the wound site of nondiabetic rats (all-trans retinoic acid or vehicle treated) had a gross and histological appearance similar to that of the all-trans retinoic acid-treated skin from the diabetic rats.
Fibronectin expression in skin from the control site, wound center, and wound edge.
On the day of wound closure, skin samples from three sites (i.e., wound center, wound edge, and beyond the original wound margin) were obtained from each animal. Organ cultures were established as described in the RESEARCH DESIGN AND METHODS section. Organ culture-conditioned medium was collected 3 days later and assessed for fibronectin expression by Western blot analysis. In the organ culture fluid from control skin (beyond the initial wound margin), there was a low (but detectable) level of immunoreactive fibronectin (Fig. 4). Organ culture fluids from both the wound center and wound edge showed increased fibronectin expression (both >3.5-fold relative to control). Fibronectin levels in organ culture fluid from wound center skin (most recently healed) were slightly higher on average than in organ culture fluid from wound margin skin (Fig. 4). Of interest, the corresponding skin samples from the four groups of animals were essentially indistinguishable from one another.
MMP expression in skin from the control site, wound center, and wound edge.
The same organ culture-conditioned medium was analyzed for MMP levels by gelatin zymography. All of the samples contained large amounts of MMP-2, and 50% of the total enzyme was in the active form (Table 3). Smaller amounts of MMP-9 were also present, with 70% of the total enzyme in the active form (Table 3). Comparing enzyme levels as a function of skin site from where the tissue was obtained demonstrated higher levels of both enzymes in tissue from the wound center and wound edge as compared with the control skin site, but the differential expression of the two enzymes was not as dramatic as was seen with fibronectin (compare data in Fig. 4 and Table 3). In comparing results from the four groups of animals, there were no obvious group-specific differences in the expression pattern for either enzyme. Thus, after wound formation and closure (6eC8 days) and subsequent incubation of the tissue for 3 days in organ culture, differences that may have been present among the four groups of animals before wounding were no longer evident in either fibronectin production or MMP elaboration.
DISCUSSION
The present study demonstrates that skin from rats with STZ-induced diabetes demonstrates histological defects (epidermal thinning, disorganization of the dermal matrix, and interstitial cell pycnosis) as compared with skin from nondiabetic control rats. These defects are evident within 8 weeks of STZ treatment and are associated with delayed healing of subsequently induced superficial abrasion wounds. Both the histological abnormalities and delayed wound healing are reduced in animals treated topically on alternative days with all-trans retinoic acid during the 8-week period after STZ administration. Results from the current study are consistent with past observations. Seifter et al. (27) demonstrated that dietary vitamin A supplementation in STZ-induced diabetic rats increased tissue strength of healed incisional wounds. In another study, all-trans retinoic acid pretreatment was demonstrated to reverse delayed wound healing in genetically diabetic mice (28). In both studies, improved wound healing was assumed to reflect more rapid formation of an extracellular matrix in the all-trans retinoic acid-treated skin.
In healthy skin, abrasion wounds typically heal without consequence. However, in individuals with diabetes, as well as in individuals with other conditions where the peripheral vasculature has been compromised, minor wounds often go on to form slow-healing ulcers with high morbidity (3,4,16,17). In light of the findings described here and in the two earlier studies (27,28), we can suggest that prophylactic use of retinoid-containing preparations might be useful in preventing the development of nonhealing skin ulcers from minor traumas in at-risk skin.
It should be kept in mind that diabetes is a progressive disease, and its consequences are often seen after years or decades. Thus, the effects of diabetes on the skin may be superimposed on the negative changes that occur as a result of the aging process itself. In this regard, the effects of diabetes on the skin may be analogous to what is seen in photodamage. Studies in hairless rodents have demonstrated that excessive ultraviolet (UV) irradiation can damage the skin connective tissue independent of the natural aging process (29,30). Clinically, however, photodamage in humans is most evident in aged skin, and we have come to see that the end result is a consequence of chronic/continuous MMP-mediated connective tissue damage (31,32) coupled with a late-stage decline in the repair process (33). The question, then, is whether retinoid treatment will be effective in the skin of individuals with diabetes as they age. Although the expression of (some) nuclear retinoid receptors changes during aging, levels of other nuclear receptors and cellular all-trans retinoic acid binding proteins appear to remain constant between young and old skin (34eC36). It is difficult to predict, therefore, based on these data, whether retinoid responsiveness in aged skin should increase, decrease, or remain unchanged as compared with young skin. It has been shown in other studies, however, that severely UV-damaged skin and chronologically aged skin both remain amenable to retinoid repair (37eC40). We can predict, based on this, that diabetic skin would also remain retinoid-sensitive during aging. Support for this comes from our own recent study in which skin biopsies from age-matched diabetic and nondiabetic individuals responded equally well to all-trans retinoic acid treatment in organ culture (22). Additional studies will need to be conducted to determine whether similar results are seen clinically. It will be critical, of course, to have age-matched nondiabetic subjects as well as diabetic duration-matched individuals with various risk profiles in such studies.
The molecular events that underlie improved wound healing in retinoid pretreated diabetic skin are not fully understood. In healthy skin, wound healing involves a well-defined sequence of responses. There is upregulation of various MMPs by resident cells (necessary for clearing of damaged tissue as well as for cell migration), deposition of a provisional matrix consisting of plasma elements as well as fibronectin synthesized by resident cells, rapid migration of keratinocytes across the surface of the provisional matrix and migration of fibroblasts into the matrix, and formation of a mature, collagen-rich matrix at the wound site (16,17). Skin at risk for chronic ulcer formation (including skin of the feet and lower limbs of individuals with diabetes) commonly demonstrates atrophic features. The dermis of such skin is characterized by the presence of fewer interstitial fibroblasts with reduced growth capacity, reduced collagen production, and increased elaboration of matrix-degrading MMPs (7eC15,21,22). Because topical retinoid treatment is known to ameliorate these features of dermal atrophy in aged and photoaged skin (31,32,38eC40), it is not unreasonable to suggest that similar changes might occur in diabetic skin. Recent studies from our laboratory indicate that this is, in fact, the case. In both diabetic human skin and diabetic rat skin, exposure to all-trans retinoic acid in organ culture resulted in decreased MMP activity and enhanced elaboration of type I procollagen (21,22). It is assumed that the beneficial retinoid effect on superficial wound healing demonstrated here is due, at least in part, to underlying improvement in dermal structure/function. Alternatively, the epidermal effects of retinoid treatment cannot be ruled out. Rapidly proliferating epidermal keratinocytes might be expected to close superficial wounds more rapidly than cells that are quiescent to begin with (41).
Skin irritation consisting of redness, dryness, and flaking occurs in many individuals after topical application of all-trans retinoic acid (42). Although the mechanism underlying the irritation response is not fully understood, retinoid effects in the epidermis are thought to be primarily responsible. Epidermal changes after topical application of all-trans retinoic acid include hyperplasia (42,43), coupled with decreased elaboration of desmosomal proteins (44eC46) and other factors that support cell-matrix attachment (47). Decreased cell-cell and cell-matrix adhesion leads to enhanced keratinocyte sloughing and barrier disruption (48,49), which in turn results in elaboration of proinflammatory cytokines such as interleukin-1 (50,51). Whereas this is an undesirable consequence of retinoid therapy (and may be particularly so in skin that is already compromised), recent studies suggest that it will be possible to separate negative epidermal effects from the beneficial effects of topical retinoid use. One possible strategy is the use of natural or synthetic retinoids that are inherently less irritating than all-trans retinoic acid. All-trans retinol (vitamin A) has been shown to have the same beneficial effects on human skin as all-trans retinoic acid, but it is less irritating to the skin than all-trans retinoic acid itself (42). Our own recent studies have shown that retinoid-induced epidermal and dermal thickening can be attained in the hairless mouse using a synthetic retinoid without inducing irritation (26). Another strategy may be to block the effects of retinoid treatment on epidermal proliferation under conditions in which dermal responses are spared. It is generally assumed that hyperplasia in the epidermis is directly or indirectly associated with irritation. Recent studies have shown that both natural and synthetic epidermal growth factor receptor antagonists inhibit retinoid-induced epidermal hyperplasia but do not inhibit retinoid effects on the dermis (43,52). Finally, it may be possible to identify nonretinoidal agents that mimic retinoid effects on the dermis without the concomitant epidermal activity of the biologically active retinoids (53). It should be possible, ultimately, to maintain the beneficial effects of retinoid treatment on the skin without the attendant epidermal disruption commonly seen.
In summary, the present study demonstrates that superficial skin wounds in diabetic rats heal more rapidly in animals that have been pretreated with a regimen of topical all-trans retinoic acid. To the extent that more rapid healing of superficial wounds counteracts the tendency to form chronic, nonhealing ulcers, topical retinoid use may have the potential to reduce formation of chronic ulcers in at-risk skin.
ACKNOWLEDGMENTS
This study was supported in part by grants DK59169 (to J.V.) and DK52391 (to M.J.S.) from the U.S. Public Health Service (USPHS), by a Veterans Affairs career development award (to M.J.S.), and by a grant from the Juvenile Diabetes Research Foundation (to M.J.S.).
MMP, matrix metalloproteinase; STZ, streptozotocin; TBS, Tris-buffered saline
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2 Department of Internal Medicine, the University of Michigan and the Ann Arbor Veterans Administration Hospital, Ann Arbor, Michigan
ABSTRACT
In the current study, rats were made diabetic with streptozotocin (STZ) and maintained for 8 weeks, during which time they were treated topically on alternative days with a solution of 0.1% all-trans retinoic acid in a vehicle of 70:30% ethanol/propylene glycol. STZ-induced diabetic rats treated with vehicle served as controls. Additional nondiabetic rats were treated with all-trans retinoic acid or vehicle in parallel. At the end of the 8-week period, rats from all four treatment groups were subjected to abrasion wound formation. Wounds healed more rapidly in vehicle-treated nondiabetic skin than in vehicle-treated diabetic skin (96% of the wound surface area closed in nondiabetic rats within 6 days vs. 41% closed in diabetic rats). Wounds in all-trans retinoic acid-treated diabetic skin healed more rapidly than wounds in vehicle-treated diabetic skin (85% of the wound surface area closed in all-trans retinoic acid-treated diabetic rats vs. 41% closed in vehicle-treated diabetic rats). At the histological level, recently healed skin from vehicle-treated diabetic rats was shown to contain a thin, wispy provisional matrix in which many of the embedded cells were rounded and some were pycnotic. In contrast, a much denser provisional matrix with large numbers of embedded spindle-shaped cells was observed in healed wounds from diabetic skin that had been pretreated with all-trans retinoic acid. The all-trans retinoic acid-treated diabetic skin was histologically similar to vehicle-treated (or all-trans retinoic acid-treated) skin from nondiabetic animals. In light of these findings, we suggest that prophylactic use of retinoid-containing preparations might be useful in preventing the development of nonhealing skin ulcers resultant from minor traumas in at-risk skin.
Diabetes is responsible for delayed or impaired wound healing, leading in many instances to chronic ulcer formation. Diabetic ulcers of the lower limbs and feet, in particular, are associated with high morbidity and often lead to amputation (1eC3). Peripheral neuropathy and peripheral vascular disease are thought to be underlying factors in diabetic wound formation (2eC6), but dermal atrophy is likely to be a contributing factor in many cases (6,7). Reduced fibroblast growth, increased expression of matrix-degrading matrix metalloproteinases (MMPs), and decreased matrix synthesis are consequences of chronic vascular disease in diabetic skin as well as in other situations where peripheral vascular disease is present (7eC15). Atrophic skin is less resistant than healthy skin to wound formation. In addition, scanty extracellular matrix production during the proliferative phase of wound repair undoubtedly contributes to poor healing (16,17).
Past studies have shown that all-trans retinoic acid has the capacity to repair photodamaged skin (18eC19). The repair response includes both increased procollagen production and decreased elaboration of tissue-destructive MMPs. In addition, there is epidermal thickening caused by retinoid-induced keratinocyte proliferation. Chronologically aged skin demonstrates the same responses to all-trans retinoic acid (20). Our own recent studies have demonstrated that diabetic skin, including skin from the lower legs of individuals at risk for ulcer formation, also responds to retinoid treatment with increased thickening, increased collagen production, and most dramatically, decreased levels of collagen-degrading MMPs (including interstitial collagenase [MMP-1] and gelatinase B [MMP-9]) (21,22). Reduction in MMP levels reflects decreased enzyme production and, especially, a reduction in enzyme activation, subsequent to an increase in TIMP-1 (tissue inhibitor of metalloproteinases-1) production in the retinoid-treated skin (22). Based on these data, we have hypothesized that prophylactic treatment of skin with all-trans retinoic acid might produce changes in the skin that would enhance wound healing and, thereby, increase resistance to chronic ulcer formation. The current studies describe efforts to test this hypothesis in an experimental model of diabetes in rats.
RESEARCH DESIGN AND METHODS
Male nude rats with a start weight of 200eC250 g were purchased from Charles River (Wilmington, MA) and acclimated for 1 week on arrival in the laboratory. Animals were randomized to control or treatment groups. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 50 mg/kg in 0.2 ml of 10 mmol/l citrate buffer, pH 5.5; Sigma, St. Louis, MO). After 48 h, blood glucose levels were assessed, and rats were included in the study if glucose concentrations were >250 mg/dl in heparinized tail vein blood (measured by glucometer). The university committee on use and care of animals approved all of the procedures used in this study.
Topical treatment with all-trans retinoic acid or vehicle.
Diabetic rats and an equal number of nondiabetic controls were divided into treatment groups. Starting 3 days after STZ administration, one group (diabetic and nondiabetic) received 100 e蘬 of a 0.1% solution of all-trans retinoic acid in 70:30% ethanol/propylene glycol, applied topically to the dorsal flank skin on both sides. Treatment was every other day for 8 weeks. A second group of diabetic and nondiabetic rats was treated with vehicle alone over the same period. All treated and control rats had ad libitum access to water and suitable rat diet. STZ-treated rats were given daily injections of a small dose of protamine zinc insulin (0.5eC3.0 units per day) as required to maintain blood glucose levels between 350 and 500 mg/dl. At the end of the 8-week treatment, four rats from each of the four groups were killed. Skin samples from the retinoid-treated or vehicle-treated sites were fixed in 10% buffered formalin and used for histology. The remainder of the rats were included in wound-healing studies.
Superficial wound formation protocol.
Rats were anesthetized with an intraperitoneal injection of ketamine/xylazine (100 mg/kg ketamine and 10 mg/kg xylazine), and the paravertebral skin was cleansed with 70% alcohol. Abrasion-type superficial wounds were made on the paravertebral dorsal skin by application of 100% acetone to the skin followed by rubbing with a coarse emery board. A circular wound area with approximate dimensions of 6 x 6 cm was made. The depth of the wound was made just to the point where oozing of fluid into the abraded tissue could be seen. The superficial wounds were designed to mimic the type of wound typically seen in human skin after minor scrapes.
Wounds were photographed using a digital camera on day 0 and on days 2, 4, and 6. Wound area was calculated as a multiple of x- and y-axes of the wound. At the time of wound closure, animals were killed by cervical dislocation. Duplicate pieces of dorsal flank skin from three different sites (i.e., wound center, wound edge, and beyond the original wound margin) were taken from each animal. One of the duplicate biopsies was immediately fixed in 10% buffered formalin and used for histology. The second piece was placed in organ culture (see below).
Organ culture.
Biopsies for organ culture were cut into pieces 2 mm on a side and incubated in wells of a 24-well dish. Five to six tissue pieces were placed in each well along with 1 ml of culture medium, consisting of Dulbecco’s modified minimal essential medium containing 200 e/ml BSA. Cultures were incubated at 37°C in an atmosphere of 5% CO2 and 95% air. Organ culture-conditioned medium collected on day 3 was used for the assessment of MMP and fibronectin levels as described below. Organ culture of rat skin has been described previously (21).
Substrate-embedded enzymography.
Substrate-embedded enzymography (zymography) (23) was used to identify and characterize MMPs. Briefly, SDS-PAGE gels were prepared from 30:1 acrylamide/bis with the incorporation of gelatin (1 mg/ml) before casting. The gels were routinely 7.5% acrylamide. Samples of organ culture fluid and molecular weight standards were electrophoresced at a constant voltage of 150 volts under nonreducing conditions. After electrophoresis, gels were removed and washed twice for 15 min in 50 mmol/l Tris buffer containing 1 mmol/l Ca2+ and 0.5 mmol/l Zn2+ with 2.5% Triton X-100. The gels were then incubated overnight in Tris buffer with 1% Triton X-100 and stained with Brilliant Blue R concentrate the following day. After destaining, zones of gelatin hydrolysis were detected as clear areas against a dark background. Zymographic images were digitized. Negative images were created and quantified by scanning densitometry. Using the digitized images, quantitative values for latent and active MMP-2 (72-kDa gelatinase A) and MMP-9 were obtained.
Western blot analysis.
Western blotting was performed as described previously (21). Briefly, a mouse polyclonal antibody to rat fibronectin was obtained from Chemicon (Temicula, CA). Equivalent amounts of organ culture-conditioned medium were resolved by SDS-PAGE (7.5%) and transferred to a nitrocellulose membrane using a Bio-Rad minitransfer blotting apparatus. Membranes were then blocked in Ca2+- and Mg2+-free Tris-buffered saline (TBS) containing 5% blotto. Membranes were then treated with the primary antibody (1:200 dilution) in TBS containing 0.5% blotto (blotting-grade nonfat dry milk; Bio-Rad, Hercules, CA) and 0.1% Tween for 1 h. After several rinses in TBS with 0.1% Tween, the membrane-bound antibody was reacted with horseradish peroxidase-conjugated rabbit anti-mouse antibody at 1:2000 dilution for 1 h. Protein-antibody complexes were detected by enhanced chemiluminescence (Cell Signaling, Beverly, MA) and visualized on light-sensitive autoradiographic film (Amersham Biotechnology, London). Images were digitized and quantified as described above with zymograms.
RESULTS
Plasma glucose values and body weights of nondiabetic and diabetic rats.
Initially, 80 hairless rats were included in the study. Half of the rats were made diabetic by giving STZ as described in the RESEARCH DESIGN AND METHODS section. The diabetic and nondiabetic rats were divided into two groups each. One set of diabetic rats was treated with 0.1% all-trans retinoic acid on the paravertebral skin, and the second set of diabetic rats was treated with vehicle only. The two groups of nondiabetic rats received the same treatment as the diabetic rats. The mean body weights and blood glucose values for each group was measured on day 1 of treatment with all-trans retinoic acid and vehicle, on day 56 of treatment (immediately after the final treatment and before wounding), and immediately before they were killed (6eC8 days later). The data are shown in Table 1.
Histological features of skin from vehicle-treated and all-trans retinoic acid-treated nondiabetic and diabetic rats.
Rats from each of the four groups were observed for changes in the physical appearance of the skin at weekly intervals throughout the study. The major difference between vehicle-treated nondiabetic and diabetic rats was that in the diabetic rats, the skin became progressively paler and more translucent over the 8-week observation period. At the time they were killed, the skin of vehicle-treated diabetic rats had a "papery thin feel." No major change in the skin from the nondiabetic rats was observed over the same period. The changes observed in the skin of vehicle-treated diabetic rats were not observed in companion rats treated with all-trans retinoic acid. Rather, the skin of all-trans retinoic acid-treated rats remained similar in appearance to that of nondiabetic rats throughout the observation period. Consistent with what has been reported (24eC26), the skin of all-trans retinoic acid-treated hairless rats (both nondiabetic and diabetic) demonstrated a degree of irritation, consisting of redness, dryness, and flaking.
Figure 1 demonstrates histological features of skin from each of the four groups of rats at 8 weeks posttreatment. Comparing vehicle-treated nondiabetic and diabetic rats, differences between the two groups included a thinner epidermis and more pycnosis in the interstitial cell population of the diabetic rats. After retinoid treatment, the epidermis of both groups was significantly thickened, and the interstitial cells of the dermis were characterized by the presence of plump, oblong, light-staining nuclei (characteristic of actively metabolizing cells). Table 2 presents epidermal thickness measurements in the four groups of rats.
Wound formation and healing.
At the end of the treatment period, circular abrasion wounds with a diameter of 35 cm2 were created in the paravertebral skin at the all-trans retinoic acid- and vehicle-treated sites. Wound healing was followed and time to wound closure measured as described above. As seen in Fig. 2, the mean wound surface area on day 0 of wounding in the vehicle-treated diabetic rats was 34.8 ± 3.4 cm2 as compared with the mean wound surface area of 37.3 ± 4.9 cm2 in vehicle-treated nondiabetic rats. By day 6 postwounding, the mean wound surface area in vehicle-treated diabetic rats was reduced to 13.7 ± 2.0 cm2, whereas the mean wound surface area in the vehicle-treated nondiabetic rats was reduced to 1.3 ± 1.4 cm2.
In the all-trans retinoic acid-treated group of diabetic rats, the original mean wound area was 34.6 ± 4.5 cm2 as compared with the original mean wound surface area of 34.8 ± 3.4 cm2 in vehicle-treated diabetic rats. By day 6, the wound surface area of the all-trans retinoic acid-treated rats was 5.1 ± 2.8 cm2 as compared with 13.7 ± 2.0 cm2 in the vehicle-treated diabetic rats. Although the data in Fig. 2 shows wound area through day 6, in some of the vehicle-treated diabetic rats, wounds had not completely healed even by day 8 (not shown). In the nondiabetic rats treated with all-trans retinoic acid, the original wound surface area was 35.4 ± 3.4 cm2. By day 6, wounds in all of the all-trans retinoic acid-treated rats had completely healed. However, because even in vehicle-treated nondiabetic rats, wounds were almost completely healed by day 6 (wound surface area of 37.3 ± 4.9 cm2 on day 0 reduced to 1.3 ± 1.4 cm2 by day 6), differences between all-trans retinoic acid- and vehicle-treated nondiabetic rats were not statistically significant.
Figure 3 demonstrates gross and histological features of vehicle-treated and all-trans retinoic acid-treated diabetic skin. Grossly, there was no visible difference in the appearance of the wounds between the two groups while they were healing, although the still-scabbed-over wound on the vehicle-treated rat is apparent at day 6, whereas the wound of the all-trans retinoic acid-treated rat has healed completely. Histologically, the appearance of the wound site in both all-trans retinoic acid-treated and vehicle-treated rats was distinguishable from control sites (beyond the wound margin) by the presence of abundant provisional matrix beneath the epidermis (lightly stained extracellular matrix in the hematoxylin and eosin-stained sections). In general, the matrix was less organized in the vehicle-treated skin than in all-trans retinoic acid-treated skin. In addition, there were few spindle-shaped or elongated cells in the provisional matrix of the vehicle-treated skin, and many of the cells had a shrunken, rounded, pycnotic appearance. In contrast, in the all-trans retinoic acid-treated skin, the majority of cells appeared to be spindle shaped (consistent with the appearance of healthy fibroblasts). Although these differences were readily apparent in many samples, there was significant variability within each group, and we were unable to accurately quantify the differences between the two groups. Skin at the wound site of nondiabetic rats (all-trans retinoic acid or vehicle treated) had a gross and histological appearance similar to that of the all-trans retinoic acid-treated skin from the diabetic rats.
Fibronectin expression in skin from the control site, wound center, and wound edge.
On the day of wound closure, skin samples from three sites (i.e., wound center, wound edge, and beyond the original wound margin) were obtained from each animal. Organ cultures were established as described in the RESEARCH DESIGN AND METHODS section. Organ culture-conditioned medium was collected 3 days later and assessed for fibronectin expression by Western blot analysis. In the organ culture fluid from control skin (beyond the initial wound margin), there was a low (but detectable) level of immunoreactive fibronectin (Fig. 4). Organ culture fluids from both the wound center and wound edge showed increased fibronectin expression (both >3.5-fold relative to control). Fibronectin levels in organ culture fluid from wound center skin (most recently healed) were slightly higher on average than in organ culture fluid from wound margin skin (Fig. 4). Of interest, the corresponding skin samples from the four groups of animals were essentially indistinguishable from one another.
MMP expression in skin from the control site, wound center, and wound edge.
The same organ culture-conditioned medium was analyzed for MMP levels by gelatin zymography. All of the samples contained large amounts of MMP-2, and 50% of the total enzyme was in the active form (Table 3). Smaller amounts of MMP-9 were also present, with 70% of the total enzyme in the active form (Table 3). Comparing enzyme levels as a function of skin site from where the tissue was obtained demonstrated higher levels of both enzymes in tissue from the wound center and wound edge as compared with the control skin site, but the differential expression of the two enzymes was not as dramatic as was seen with fibronectin (compare data in Fig. 4 and Table 3). In comparing results from the four groups of animals, there were no obvious group-specific differences in the expression pattern for either enzyme. Thus, after wound formation and closure (6eC8 days) and subsequent incubation of the tissue for 3 days in organ culture, differences that may have been present among the four groups of animals before wounding were no longer evident in either fibronectin production or MMP elaboration.
DISCUSSION
The present study demonstrates that skin from rats with STZ-induced diabetes demonstrates histological defects (epidermal thinning, disorganization of the dermal matrix, and interstitial cell pycnosis) as compared with skin from nondiabetic control rats. These defects are evident within 8 weeks of STZ treatment and are associated with delayed healing of subsequently induced superficial abrasion wounds. Both the histological abnormalities and delayed wound healing are reduced in animals treated topically on alternative days with all-trans retinoic acid during the 8-week period after STZ administration. Results from the current study are consistent with past observations. Seifter et al. (27) demonstrated that dietary vitamin A supplementation in STZ-induced diabetic rats increased tissue strength of healed incisional wounds. In another study, all-trans retinoic acid pretreatment was demonstrated to reverse delayed wound healing in genetically diabetic mice (28). In both studies, improved wound healing was assumed to reflect more rapid formation of an extracellular matrix in the all-trans retinoic acid-treated skin.
In healthy skin, abrasion wounds typically heal without consequence. However, in individuals with diabetes, as well as in individuals with other conditions where the peripheral vasculature has been compromised, minor wounds often go on to form slow-healing ulcers with high morbidity (3,4,16,17). In light of the findings described here and in the two earlier studies (27,28), we can suggest that prophylactic use of retinoid-containing preparations might be useful in preventing the development of nonhealing skin ulcers from minor traumas in at-risk skin.
It should be kept in mind that diabetes is a progressive disease, and its consequences are often seen after years or decades. Thus, the effects of diabetes on the skin may be superimposed on the negative changes that occur as a result of the aging process itself. In this regard, the effects of diabetes on the skin may be analogous to what is seen in photodamage. Studies in hairless rodents have demonstrated that excessive ultraviolet (UV) irradiation can damage the skin connective tissue independent of the natural aging process (29,30). Clinically, however, photodamage in humans is most evident in aged skin, and we have come to see that the end result is a consequence of chronic/continuous MMP-mediated connective tissue damage (31,32) coupled with a late-stage decline in the repair process (33). The question, then, is whether retinoid treatment will be effective in the skin of individuals with diabetes as they age. Although the expression of (some) nuclear retinoid receptors changes during aging, levels of other nuclear receptors and cellular all-trans retinoic acid binding proteins appear to remain constant between young and old skin (34eC36). It is difficult to predict, therefore, based on these data, whether retinoid responsiveness in aged skin should increase, decrease, or remain unchanged as compared with young skin. It has been shown in other studies, however, that severely UV-damaged skin and chronologically aged skin both remain amenable to retinoid repair (37eC40). We can predict, based on this, that diabetic skin would also remain retinoid-sensitive during aging. Support for this comes from our own recent study in which skin biopsies from age-matched diabetic and nondiabetic individuals responded equally well to all-trans retinoic acid treatment in organ culture (22). Additional studies will need to be conducted to determine whether similar results are seen clinically. It will be critical, of course, to have age-matched nondiabetic subjects as well as diabetic duration-matched individuals with various risk profiles in such studies.
The molecular events that underlie improved wound healing in retinoid pretreated diabetic skin are not fully understood. In healthy skin, wound healing involves a well-defined sequence of responses. There is upregulation of various MMPs by resident cells (necessary for clearing of damaged tissue as well as for cell migration), deposition of a provisional matrix consisting of plasma elements as well as fibronectin synthesized by resident cells, rapid migration of keratinocytes across the surface of the provisional matrix and migration of fibroblasts into the matrix, and formation of a mature, collagen-rich matrix at the wound site (16,17). Skin at risk for chronic ulcer formation (including skin of the feet and lower limbs of individuals with diabetes) commonly demonstrates atrophic features. The dermis of such skin is characterized by the presence of fewer interstitial fibroblasts with reduced growth capacity, reduced collagen production, and increased elaboration of matrix-degrading MMPs (7eC15,21,22). Because topical retinoid treatment is known to ameliorate these features of dermal atrophy in aged and photoaged skin (31,32,38eC40), it is not unreasonable to suggest that similar changes might occur in diabetic skin. Recent studies from our laboratory indicate that this is, in fact, the case. In both diabetic human skin and diabetic rat skin, exposure to all-trans retinoic acid in organ culture resulted in decreased MMP activity and enhanced elaboration of type I procollagen (21,22). It is assumed that the beneficial retinoid effect on superficial wound healing demonstrated here is due, at least in part, to underlying improvement in dermal structure/function. Alternatively, the epidermal effects of retinoid treatment cannot be ruled out. Rapidly proliferating epidermal keratinocytes might be expected to close superficial wounds more rapidly than cells that are quiescent to begin with (41).
Skin irritation consisting of redness, dryness, and flaking occurs in many individuals after topical application of all-trans retinoic acid (42). Although the mechanism underlying the irritation response is not fully understood, retinoid effects in the epidermis are thought to be primarily responsible. Epidermal changes after topical application of all-trans retinoic acid include hyperplasia (42,43), coupled with decreased elaboration of desmosomal proteins (44eC46) and other factors that support cell-matrix attachment (47). Decreased cell-cell and cell-matrix adhesion leads to enhanced keratinocyte sloughing and barrier disruption (48,49), which in turn results in elaboration of proinflammatory cytokines such as interleukin-1 (50,51). Whereas this is an undesirable consequence of retinoid therapy (and may be particularly so in skin that is already compromised), recent studies suggest that it will be possible to separate negative epidermal effects from the beneficial effects of topical retinoid use. One possible strategy is the use of natural or synthetic retinoids that are inherently less irritating than all-trans retinoic acid. All-trans retinol (vitamin A) has been shown to have the same beneficial effects on human skin as all-trans retinoic acid, but it is less irritating to the skin than all-trans retinoic acid itself (42). Our own recent studies have shown that retinoid-induced epidermal and dermal thickening can be attained in the hairless mouse using a synthetic retinoid without inducing irritation (26). Another strategy may be to block the effects of retinoid treatment on epidermal proliferation under conditions in which dermal responses are spared. It is generally assumed that hyperplasia in the epidermis is directly or indirectly associated with irritation. Recent studies have shown that both natural and synthetic epidermal growth factor receptor antagonists inhibit retinoid-induced epidermal hyperplasia but do not inhibit retinoid effects on the dermis (43,52). Finally, it may be possible to identify nonretinoidal agents that mimic retinoid effects on the dermis without the concomitant epidermal activity of the biologically active retinoids (53). It should be possible, ultimately, to maintain the beneficial effects of retinoid treatment on the skin without the attendant epidermal disruption commonly seen.
In summary, the present study demonstrates that superficial skin wounds in diabetic rats heal more rapidly in animals that have been pretreated with a regimen of topical all-trans retinoic acid. To the extent that more rapid healing of superficial wounds counteracts the tendency to form chronic, nonhealing ulcers, topical retinoid use may have the potential to reduce formation of chronic ulcers in at-risk skin.
ACKNOWLEDGMENTS
This study was supported in part by grants DK59169 (to J.V.) and DK52391 (to M.J.S.) from the U.S. Public Health Service (USPHS), by a Veterans Affairs career development award (to M.J.S.), and by a grant from the Juvenile Diabetes Research Foundation (to M.J.S.).
MMP, matrix metalloproteinase; STZ, streptozotocin; TBS, Tris-buffered saline
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