MDI 301, a non-irritating retinoid, induces changes in human skin that underlie repair
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Previous studies have demonstrated that all-trans retinoic acid (RA) increases collagen production and decreases matrix metalloproteinase (MMP) activity in organ-cultured human skin. Decreased MMP activity is associated with up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1). These changes are accompanied by a hyperplastic response in the epidermis. Here we show that a synthetic picolinic ester-substituted retinoid (designated as MDI 301) has comparable effects to those of RA in regard to these activities. What makes these findings of interest is that RA also stimulates elaboration of several pro-inflammatory cytokines and up-regulates leukocyte adhesion molecules in organ-cultured skin. MDI 301 does not induce such changes or is much less active. In a past study we showed that while RA was irritating to the skin of topically treated hairless mice, MDI 301 was essentially non-irritating under the same conditions [Varani et al. (2003) Arch. Dermatol Res 295:255–262]. Taken in conjunction with the findings from the past study, the present data suggest that MDI 301 will be similar to RA in capacity to repair damaged skin, but will be effective under conditions that are not irritating. These findings, thus, suggest that retinoid efficacy and clinically relevant irritancy are not inextricably linked. Potential for efficacy under conditions in which irritation is not observed is a strong rationale for further development of MDI 301 as a skin-repair agent.
KeywordsAll trans retinoic acid MDI 301 Skin Type-I procollagen Matrix metalloproteinase Tissue inhibitor of metalloproteinases Cytokines Leukocyte adhesion molecules
Topical application of all-trans retinoic acid (RA) improves the appearance of skin damaged as a consequence of chronic exposure to ultraviolet radiation from the sun (photoaging) [17, 33]. Retinoid use also improves the appearance of chronologically aged skin . In both photoaging and natural aging, there is a loss of intact collagen and an increase in fragmented collagen in the skin [10, 26, 32]. RA treatment reverses the increased expression of connective tissue-degrading matrix metalloproteinases (MMPs) that are responsible for collagen degradation in the skin [7, 8] and concomitantly stimulates new collagen synthesis [13, 32]. These cellular events lead to changes in connective tissue structure that are visible at the histological level and, presumably, underlie the improved appearance. This has been convincingly demonstrated in both clinical studies and experimental animal models [16, 17, 18, 19, 33]. Given the ability of RA to reduce collagen damage and induce new collagen synthesis, it is not surprising that topical retinoid use not only improves the appearance of damaged skin but also results in better function. Specifically, a number of past studies have demonstrated that RA-pretreatment improves healing of wounds that subsequently occur in skin that has been damaged as a result of aging/photoaging or as a consequence of diabetes or chronic corticosteroid use [2, 20, 25, 34, 36].
A consequence of topical retinoid use is skin irritation. Irritated skin is characterized by redness, dryness and flaking at the treated site. At the histological level, one sees a perivascular accumulation of mononuclear cells, with neutrophils and monocyes scattered throughout the dermis and occasional micro-abscesses in the dermis or epidermis . All-trans retinol (vitamin A), the parent compound of RA, tends to be less irritating than RA in most cases, but even with this agent, significant irritation is observed in many individuals . Likewise, skin irritation is also a complication with synthetic agents currently on the market . Irritation is a major cause of non-compliance among retinoid users. In addition, excessive irritation may counteract the beneficial effects of topical use.
It has been assumed in the past that retinoid efficacy and retinoid irritation could not be separated. Recent studies, however, cast doubt on this. We demonstrated in a recent study that an ester-substituted derivative of 9-cis RA (termed MDI 301) was as efficacious as RA or 9-cis RA in stimulating epidermal hyperplasia and dermal thickening when applied topically to the skin of hairless mice . Unlike RA, the synthetic agent appeared to be completely non-irritating to the rodent skin following topical use. Although hairless mice are commonly used as model for retinoid responses in skin, whether or not MDI 301 will be as effective as RA in repairing aged or photoaged human skin can only be established with human skin, itself. As a way to begin addressing this issue, MDI 301 and RA were compared for effects on human skin cells (dermal fibroblasts and epidermal keratinocytes) in monolayer culture and for effects on human skin in organ culture. The data presented here demonstrate that in both monolayer and organ culture models, MDI 301 was able to stimulate events that underlie skin repair. Of interest, MDI-301 did not up-regulate pro-inflammatory cytokines including interleukin-1β (IL-1β), IL-6, IL-8, or macrophage chemotactic peptide-1 (MCP-1) while these cytokines were induced by RA. Concomitantly, MDI 301 was less effective than RA in inducing expression of intercellular adhesion molecule-1 (ICAM-1) and E-Selectin in the microvasculature of organ-cultured skin.
Materials and methods
MDI 301 is a 9-cis RA derivative in which the terminal carboxylic acid group has been replaced by a picolinic ester. MDI 301 was synthesized as described in the original patent (US Patent 5,837,728; Molecular Design International; Memphis, TN) and our previous report . RA was purchased from Sigma Chemical Company (St. Louis, MO). Both retinoids were stored at −80°C protected from light. For use in cell culture and organ culture, the two retinoids were prepared in DMSO at 20 mg/ml, aliquoted and frozen at −80°C protected from light. The DMSO stocks were stable for at least 1 year. At the time of use, the retinoids were diluted directly in culture medium at a final working solution of no greater than 5 μg/ml. At this concentration, the final amount of DMSO in the medium was 0.025 μl/ml. At this level, DMSO had no detectable effects on cell function.
Human skin organ culture
Replicate 2-mm punch biopsies were obtained from hip skin of volunteers less than 70 years of age. The participation of human subjects in this study was approved by the University of Michigan Institutional Review Board and all subjects provided written informed consent prior to their inclusion in the study. The punch biopsies were incubated in wells of a 24 well dish (one tissue piece per 250 μl of culture medium). Culture medium consisted of Keratinocyte Basal Medium (KBM) (Cambrex Biologicals, Walkersville, MD). KBM is a modification of MCDB-153 medium. For our purposes, it was supplemented with calcium chloride to a final Ca2+ concentration of 1.4mM. One well was left as control while the others were treated with RA or MDI 301 (0.5–1 μg/ml). Fresh culture medium was provided at 2-day intervals. Organ culture-conditioned medium was saved for assessment of type-I procollagen, MMPs (MMP-2 and -9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) as described below. The same culture fluid was also analyzed for IL-1β, IL-6, IL-8 and MCP-1 as described below. At the end of the incubation period (normally at day-8), organ-cultured tissue was fixed in 10% buffered formalin and embedded in paraffin. Five-μm thick sections were cut and stained with hematoxylin and eosin. Representative sections of each biopsy were selected for histological evaluation and photographed. Epidermal thickness measurements were made from these tissue sections. In certain experiments, tissue was frozen in Optimal Cutting Temperature (OCT) medium and used for immunohistology. Human skin in organ culture has been extensively used in the past to help understand how RA affects skin structure and function. The organ culture protocol used here is virtually identical to that described in a past report .
Substrate embedded Enzymography (zymography) was used in these studies to assess levels of latent and active MMP-2 and MMP-9 in organ culture and cell culture fluids. As described previously , SDS-PAGE gels were prepared with the incorporation of gelatin (1 mg/ml) at the time of casting. After electrophoresis and overnight incubation, zones of hydrolysis were identified and quantified. Values for latent and active MMP-2 and MMP-9 bands were obtained.
Culture fluids were assayed for type-I procollagen by enzyme-linked immunosorbant assay (ELISA) (Pan Vera Corp., Madison, WS) as described previously . Type-I procollagen has the terminal peptide sequences that are present at synthesis and, therefore, is a measure of ongoing collagen synthesis.
Culture fluids were assayed for TIMP-1 by ELISA using a commercially available assay kit (R&D Systems, Mpls. MN) .
Levels of IL-1β, IL-6, IL-8 and MCP-1 were quantified. Individual ELISA kits were used for IL-6, IL-8 and MCP-1 while IL-1β was quantified as part of a multiplex assay (BioSource International Inc., Camarillo, CA).
Organ-cultured tissue was fixed in OCT medium after two days of incubation. The frozen tissue was sectioned and stained for expression of ICAM-1 or E-Selectin. The antibody to ICAM-1 was a mouse monoclonal IgG1 antibody (HA58) from BD-Pharmingen (San Jose, CA). The antibody to E-Selectin was also a mouse monoclonal IgG1 antibody (clone 7A9) obtained from the American Type Culture Collection (ATCC). The sections were stained by the immunoperoxidase method and the reaction product was visualized using diaminobenzadine as the chromogenic substrate. Immunostained sections were examined by light microscopy.
Human epidermal keratinocytes and human dermal fibroblasts in monolayer culture
Normal human epidermal keratinocytes and normal human dermal fibroblasts were isolated from 2-mm punch biopsies of skin as described previously . Primary and early passage keratinocytes were maintained in Keratinocyte Growth Medium (KGM) (Cambrex Inc.). KGM consists of the same basal medium as KBM but is further supplemented with a mixture of growth factors including 0.1 ng/ml EGF, 0.5 μg/ml insulin, and 2% bovine pituitary extract. In addition to using low-passage keratinocytes, we also used the HaCat line of immortalized human epidermal keratinocytes in some experiments . The immortalized keratinocytes were handled exactly as low-passage keratinocytes.
Fibroblasts were obtained from the same tissue and grown in monolayer culture using Dulbecco’s Modified Minimal Essential Medium supplemented with nonessential amino acids and 10% fetal bovine serum (DMEM-FBS). Both keratinocytes and fibroblasts were maintained at 37°C in an atmosphere of 95% air and 5% CO2. Cells were subcultured by exposure to trypsin/ethylenediamine tetraacetic acid (EDTA) and used at passage 2–3.
Keratinocyte adhesion assay
Keratinocytes adhesion was assessed as described previously . Briefly, cells were plated in wells of a 24-well dish at 5 × 104 cells per well in growth medium and treated with RA or MDI 301. At the end of the incubation period, cells were exposed to a solution of 0.05% trypsin/0.02% EDTA and incubated for 15 min. At the end of the incubation period, non-attached cells from each well were harvested and counted. Fresh trypsin/EDTA solution was added and the cells re-incubated until all had detached. Cells recovered in the final incubation were also counted. The percentage of cells detached at the intermediate time-point was determined from these values.
Fibroblast survival assay
Fibroblast survival was assessed as described previously . Briefly, cells were seeded at 5 × 104 cells per well in DMEM-FBS (24-well plate) and allowed to attach overnight. Following this, cells were washed and put into assay buffer (KBM). KBM consists of the same basal medium as KGM but is not supplemented with growth factors. Cells were treated with different concentrations of MDI 301 (or RA), and cell numbers were assessed three days later. For counting, cells were released from the substrate with trypsin/EDTA and enumerated using a particle counter (Coulter Electronics, Hialeah, FL).
Histological features of retinoid-treated human skin in organ culture: comparison of RA and MDI 301
In addition to examining organ-cultured human skin for changes in epidermal structure, effects of retinoid treatment on dermal features were also examined. Neither agent induced observable histological changes in the connective tissue structure over the 8-day period (Fig. 1). This is not surprising since previous studies have shown that several weeks to months are required before improvement in collagen structure is observed with topical application of RA to human skin [16, 33].
MMP-2, MMP-9 and TIMP-1 in organ cultures of retinoid-treated human skin: comparison of RA and MDI-301
Collagen production in retinoid-treated human skin in organ culture: comparison of RA and MDI 301
Effects of RA and MDI 301 on cytokine elaboration and leukocyte adhesion molecule expression in organ-cultured skin
Effects of RA and MDI 301 on keratinocyte adhesive function in monolayer culture
Effects of RA and MDI 301 on fibroblast survival
RA and the other biologically active derivatives of vitamin A (all-trans retinol) have hormone-like activities in the skin. Where the skin has become hyperplastic (for example, in psoriasis and in certain malignancies), retinoids effectively suppress proliferation [6, 9]. On the other hand, where skin cell proliferation has slowed (for example, in aged skin), retinoid stimulate growth and result in epidermal thickening . Retinoid stimulatory effects also extend to the dermis, where suppression of the major collagen-degrading enzymes [7, 8] and stimulation of procollagen synthesis [13, 18, 19] improve appearance [16, 17, 33] and improve the healing of wounds that subsequently occur [2, 20, 25, 34]. In spite of the beneficial effects, the capacity of RA to irritate the skin makes it less than desirable for many users. At one time it was believed that retinoid efficacy and retinoid irritation could not be separated. This may not be entirely correct. Others have shown that vitamin A mediates events similar to RA but is less irritating in human skin . It has also been shown that by carefully controlling the dosage, irritation can be mitigated under conditions in which beneficial activities remain . In a past study we showed that a synthetic, ester-substituted retinoid (i.e., MDI 301) was similar to RA in its ability to induce epidermal hyperplasia in hairless mice and to induce a thickening of the collagen band immediately beneath the epidermis . These changes in skin structure were seen in spite of the fact that unlike RA, there was essentially no irritation in the skin of the MDI 301-treated animals. Although hairless rodents are commonly used as a model for retinoid responses in skin, rodent skin is significantly different from human skin, and it is important to establish beneficial effects in human skin.
Here we show that human skin in organ culture responds to MDI 301 with similar responses as observed with RA. The two retinoids induced a comparable degree of hyperplasia in the epidermis. At supra-optimal concentrations, both retinoids induced abnormalities in the upper epidermis (hyperkeratosis, acantholysis and partial separation of the differentiating cells from the basal cells). Along with the reduced epidermal cohesiveness observed in whole skin, treatment of keratinocytes with either RA or MDI 301 in monolayer culture resulted in decreased cell-substrate adhesion. The two retinoids were also comparable in stimulating type-I procollagen synthesis and inhibiting MMP function. MMP reduction was accompanied by increased production of TIMP-1 with both agents. Based on these data, we suggest that MDI 301 will ultimately prove to be an effective skin-repair agent.
In addition to examining events related to efficacy, we also compared the two retinoids for effects on cellular responses that may contribute to irritation. MDI-301 was clearly less active than RA in inducing production of pro-inflammatory cytokines and in up-regulating leukocyte adhesion molecules (events closely tied to skin inflammation). What accounts for these differences, however, is unknown. Retinoid irritation is a complex and not completely understood problem. It has been suggested that irritation is, in some manner, connected to epidermal hyperplasia. Rapidly proliferating keratinocytes elaborate large amounts of pro-inflammatory cytokines [22, 23]. In addition to inducing hyperplasia, retinoid treatment also alters the pattern of differentiation in the epidermis and reduces barrier function [5, 11, 14]. The loss of barrier function rather than hyper-proliferation per se may be directly responsible for promoting pro-inflammatory events . Our data do not contradict these suggestions. On the other hand, our findings strongly imply that hyperplasia and irritation are not inextricably linked. Even changes in epidermal barrier function (to the extent that the observations made here—altered differentiation, separation of upper epidermal layers from the basal layer and decreased adhesiveness in vitro—reflect alterations in the epidermal barrier) appear not to be directly responsible for irritation. In all these regards, the effects of RA and MDI 301 were comparable. Yet RA stimulated the production of pro-inflammatory cytokines under conditions in which MDI-301 did not.
Although there was no observable difference in the epidermal response to the two retinoids (either in organ culture or with keratinocytes in monolayer culture) that might suggest a basis for the difference in irritancy, the two retinoids were significantly different in their capacity to prevent fibroblast lysis under low-Ca2+ conditions (ED50 = 0.3 μg/ml with RA as compared with 2.0 μg/ml with MDI 301). Whether this difference is related, in any way, to the difference in irritancy is not known. Of interest in this regard, it has been demonstrated previously that certain detergents such as sodium lauryl sulfate also preserve fibroblast viability under low-Ca2+ conditions . Since retinoid has a detergent-like structure (polar head group connected to a hydrophobic, fatty acid tail) and have been shown to function in a detergent-like manner , perhaps irritation is a function of the detergent property. If this turns out to be the case, then it may be that MDI 301, with its ester substitution for the free acid group, is inherently less detergent-like than RA.
Another possibility is that the lower capacity of the ester-substituted retinoid to induce irritation reflects altered penetrability. In a past study comparing RA and MDI-301 for anti-cancer activity, it was noted that both retinoids induced epidermal hyperplasia in nude mice following intraperitoneal injection . While there was no significant skin irritation in either group of mice, mice injected with either RA or MDI 301 developed a comparable degree of splenomegaly with enlarged germinal centers (indicative of a systemic inflammatory response).
Regardless of the underlying mechanism, it is clear from our recent study that MDI 301 is inherently less irritating that RA when used topically in hairless rodents . The findings presented here demonstrate that MDI-301 is comparable to RA in its ability to induce events that underlie repair of damaged skin in human skin, but unlike RA, does not induce elaboration of pro-inflammatory cytokines or up-regulate expression of leukocyte adhesion molecules in the skin. Our studies, therefore, (1) suggest that repair efficacy and irritation are not inextricably linked; (2) provide insight into possible mechanisms of irritation; and (3) provide a rationale for further development of MDI 301 as a skin-repair agent.
This study was supported in part by grants AR49621 and GM77724 from the USPHS.
- 6.Esgleyes-Ribot T, Chandraratna RA, Lew-Kaya DA, Sefton J, Duvic M (1987) Response of psoriasis to a new topical retinoid, AGN 190168. J Am Acad Dermatol 30:581–590Google Scholar
- 12.Griffiths CE, Kang S, Ellis CN, Kim KJ, Finkel LJ, Ortiz-Ferrer LC, White GM, Hamilton TA, Voorhees JJ (1995) Two concentrations of topical tretinoin (retinoic acid) cause similar improvement of photoaging but different degrees of irritation. A double-blind, vehicle-controlled comparison of 0.1% and 0.025% tretinoin cream. Arch Dermatol 131:1037–1044PubMedCrossRefGoogle Scholar
- 15.Kang S, Duell EA, Fisher GJ, Datta SC, Wang Z-Q, Reddy AP, Tavakkol A, Voorhees JJ (1995) Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid-binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation. J Invest Dermatol 105:549–556PubMedCrossRefGoogle Scholar
- 18.Kligman LH (1986) Effects of all-trans-retinoic acid on the dermis of hairless mice. J Am Acad Dermatol 15:779–785 884–777Google Scholar
- 32.Varani J, Warner RL, Gharaee-Kermani M, Phan SH, Kang S, Chung J, Wang ZQ, Datta SH, Fisher GJ, Voorhees JJ (2000) Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol 114:480–486PubMedCrossRefGoogle Scholar