Statistical analysis - MATERIALS AND METHODS

B. MATERIALS AND METHODS

5. Statistical analysis

A paired Student’s t-test was used to determine statistical significance of the differences between the staining patterns using the SPSS 11.0 statistics program (SPSS, Inc., Chicago, IL, USA). All P values were two-tailed, and differences were considered significant when the P-value was less than 0.05. Summary data are expressed as the mean ± standard deviation (SD).

III. RESULTS

1. Clearance of AKs

The ALA-PDT was tolerated in all patients. During the treatment, variable degrees of erythema and edema occurred, but resolved within a few days. Some patients reported mild to moderate pain, but interruption of procedure or topical anesthesia were not required.

Initially, 23AKs were detected among the patients. One month after 2 sessions of PDT, 19 lesions from 11 patients showed a complete clinical and histological response (remission rate:

82.6%) (Fig. 1A, B). Clinically, there was no significant scarring or pigmentary changes after treatment (Fig. 1A). Among four patients with persistent lesions, one patient with two lesions received two additional PDT treatments, the AKs in the follow up biopsy resolved. The other two patients underwent cryotherapy twice with liquid nitrogen, and the lesions cleared (Table 1).

Table 1. Baseline characteristics of patients and course of treatment of AKs

pre- PDT post- PDT

Fig. 1. Clearance of AK after ALA-PDT. Patient 9 with actinic keratosis on the nose showed complete clinical clearance of lesion (A). The atypical keratinocytes in the epidermis were all disappeared after treatment in the same patient (H&E, x 200) (B).

2. Mean epidermal thickness and inflammatory infiltrates

The pre-treatment specimens showed thick epidermis and a large number of infiltrating cells in the dermis. However, after treatment, the mean epidermal thickness and the inflammatory infiltrate were significantly reduced (Fig. 2 A, B).

pre- PDT post- PDT

Fig. 2. Decreased epidermal thickness and dermal inflammatory infiltrate after ALA-PDT.

The epidermal thickness and numerous dermal inflammatory infiltrate decreased after PDT (patient 2, H&E, x 200) (A). The mean epidermal thickness in photodamaged facial skin was significantly reduced from 146.10 ±173.20 um to 75.26±29.70 um after treatment. And in the semi-quantitative analysis, the dermal inflammatory infiltrate also significantly decreased (B).

(*p<0.00, **p<0.02) A.

B. * **

3. Total collagen volume

After PDT, almost all biopsy specimens showed impressive collagen deposition in the upper dermis by H&E staining that was more clearly demonstrated by Masson-trichrome staining (Fig.

3 A, B). The differences in staining density were significant (Fig. 3 C).

pre- PDT post- PDT Masson-trichrome (B) stained specimens demonstrated up-regulated collagen fibers in the upper dermis after PDT (patient 7, x 200). Staining with Masson-trichrome demonstrated a 10.49

±9.93 % average increase of collagen in staining density in the post-treatment than in the pre-treatment specimens (p<0.00) (C).

4. Procollagen type I and type III

The expression of collagen precursors was also increased. Staining with procollagen type I and type III antibodies demonstrated an 18.03±16.41% and 11.50±9.97% average increase in their staining density, respectively (Fig. 4 A-C).

pre- PDT post- PDT type III (B) procollagen expression was increased in skin sections after ALA-PDT (patient 10, lesion 1, x 200). The average increase was significantly different (C). (*p<0.05)

5. TGF-ββββ and TββββR II

Fig. 5. Increased expression of TGF-ββββ and TββββR II in the epidermis after treatment. TGF-β

(patient 3, x 200) (A) and TβR II (patient 14, x 200) (B) was more expressed in the epidermis after ALA-PDT. The mean staining with TGF-β and TβR II antibodies demonstrated a 23.57±15.16% and 6.61±9.56% average increase in staining density, respectively (C). (*p<0.05)

In one patient with two AK lesions that underwent two additional PDT sessions, the mean expression of TGF-β, TβR II, procollagen I, procollagen III, and total collagen increased more proportional to the number of ALA-PDT sessions. (Fig. 6)

Fig. 6. Accumulative effect of ALA-PDT on the expression of TGF-ββββ, TββββR II, procollagen I, procollagen III, and total collagen volume. The mean expression of TGF-β, TβR II, procollagen I, procollagen III, and total collagen volume was more and more increased with sessions of ALA-PDT (patient 8).

TGF-β TβR II

6. Elastotic material

In the H&E stained sections, a marked elastotic mass was visible in the papillary and reticular dermis of the pre-PDT specimens that were pathognomonic for photoaging. After ALA-PDT, there was a tendency for decrease of these elastotic masses compared to the pre-treatment specimens. For patient 8 who received four treatment sessions, the change was more pronounced (Fig. 7A). Verhoeff’s elastic stain demonstrated that after the PDT, the thickened and amorphous elastotic materials disappeared and were restored to more normal horizontally arranged fibers in the dermis (Fig. 7B).

pre- PDT post- PDT

Fig. 7. Improvement of solar elastosis by ALA-PDT. After ALA-PDT, solar elastosis was improved (patient 8, lesion 1, H&E, x 200) (A), and elastin fiber reorganization was more clearly presented with Verhoeff’s stain (B) (same patient, x 400).

7. Fibrillin-1 and tropoelastin

In the dermis, expression of fibrillin-1 and tropoelastin were mainly co-localized with elastotic material, found in the epidermis to a lesser extent. After treatment, the immunoreactivity of fibrillin-1 and tropoelastin was reduced along with resolution of the solar elastosis; the epidermal expression also decreased (Fig. 8A-C).

pre- PDT post- PDT

Expression of fibrillin-1 and tropoelastin

0 0.5 1 1.5 2 2.5 3 3.5

pre-PDT post-PDT

Mean target antigen staining semi-quantitative analysis

Fibrillin-1 Tropoelastin

C * *

Fig. 8. Decreased fibrillin-1 and tropoelastin after ALA-PDT. The fibrillin-1 (A) and tropoelastin (B) expression decreased in the dermis after treatment (patient 8, lesion 2, x 200).

The mean difference was 1.32±0.90 and 0.56±0.58 in the semi-quantitative analysis after ALA-PDT (C). (*p<0.05)

8. MMPs and TIMP

I also investigated changes in the MMPs, degradating enzymes of ECM, and TIMP-1, an inhibitor of MMPs. The expression of MMP-1(Fig. 9A), MMP-3(Fig. 9B), and MMP-12 (Fig.

9C) tended to decrease after treatment. The immunoreactivity of TIMP-I was minimal when evaluated by our method.

pre- PDT post- PDT

Expression of MMP-1,-3,-12

0 0.5 1 1.5 2 2.5

pre-PDT post-PDT Mean target antigen staining semi-quantitative analysis MMP-1 MMP-3 MMP-12

Fig. 9. Decreased expression of MMP1, 3, and 12 after ALAPDT. The degree of MMP1, -3, and -12 expression reduced after ALA-PDT (A-C). The differences were 0.60±0.91, 0.64±0.91, and 0.68±1.14 in the semi-quantitative analysis, respectively (D). (*p<0.05)

* *

IV. DISCUSSION

Topical ALA-PDT was originally used for superficial non-melanoma skin cancers and their precursors (Fritsch et al., 1998). However other benign diseases, such as acne vulgaris, sebaceous gland hyperplasia, and hidradenitis suppurativa have been shown to improve with this treatment (Szeimies et al., 2002). Moreover, previous studies have demonstrated the effectiveness of ALA-based PDT treatments using a variety of lasers and light sources for photorejuvenation (Table 2). However, most prior data has been based on clinical observation, without histopathological confirmation. The results of the present study provide histopathological evidence for photorejuvenation with ALA-PDT in patients with AKs.

Table. 2 Summary of recent studies of ALA-PDT in photorejuvenation improvement skin appearance by reducing wrinkling, shallowness, and dyspigmentation

Significantly greater improvement in global photodamage, mottled pigmentation, and fine lines in the ALA-PDT-IPL group than

An increase in type I collagen fibers was seen in the ALA-PDT-IPL group than treatment skin roughness, mottled hyperpigmentation, and telangectasias, and clearance rate of AK

Upregulation of collagen production was shown with increase in procollagen type I and III mRNA

IPL, intense pulsed light ; LED, light emitting diodes; PDL, pulsed dye laser; * Split-face study

In photoaged skin, a decrease in type I and III collagen is more prominent than in intrinsic aged skin. Ultraviolet induces MMPs that can degrade collagen in human skin. MMP-1 initiates cleavage of type I and III collagen, and the damaged collagen can be further degraded by MMP-3 and MMP-9 (Sternlicht and Werb, 2001). Chronic UV exposure likely causes the accumulation of MMP-mediated collagen damage; eventually the total collagen volume in the dermis will decline as a result. In the present study, the expression of MMP-1 and MMP-3 was found to be decreased one month after ALA-PDT.

In addition to destruction of mature collagen, UV irradiation impairs synthesis of new collagen, which is reflected by the down-regulation of type I and type III procollagen gene expression (Fisher et al., 2000). In addition, the TGF-β/Smad pathway is impaired by down-regulation of TβRII and contributes to the pathology (Quan et al., 2004). TGF-β is a major cytokine that stimulates fibroblast proliferation important to enhancing collagen synthesis in the dermis (Inagaki et al., 1994). TGF-β initially binds to cell surface receptors, the TGF-β type I receptor and TβRII. Then the activated complex interacts with intracellular signal transducer Smad proteins that conveys TGF-β signaling (Quan et al., 2002). However, UV irradiation

down-regulates TβRII, which eventually decreases type I procollagen expression in human skin (Quan et al., 2004). In the present study, the expression of type I and III procollagen, the precursors of collagen, were constitutionally increased after treatment, which reflect the increased synthesis of dermal collagen. In addition, the immunoreactivity of TGF-β as well as TβR II was significantly increased after treatment, which indicates that they contribute to the reconstitution of the ECM.

The increase of dermal collagen, TGF-β and TβR II expression was more significant in patients who received two more additional PDT treatments. This suggested a cumulative effect of the

ALA-PDT on photorejuvenation.

Another prominent feature of photoaged skin is the accumulation of dystrophic elastotic material in the reticular dermis, known as solar elastosis (Montagna et al., 1989). Elastic fibers consist of a central core of elastin and surrounding fibrillin-rich microfibrils (Mecham RP, 1991).

Increased tropoelastin gene expression, both in the epidermal keratinocytes and fibroblasts of human skin, has been shown in vivo with UVB irradiation (Seo et al., 2001); this may be the process by which elastotic materials accumulate in photodamaged skin. Moreover, in the reticular dermis of photodamaged skin, an increased expression and deposition of fibrillin were demonstrated (Bernstein et al., 1994). In addition to the increased production of tropoelastin and fibrillin, degradation of elastic fibers contributes to the solor elastosis. MMP-12, the active degradating enzyme against elastin, was reported to co-localize with the elastotic material in photoaged skin, and the expression of MMP-12 mRNA and protein was induced by UV irradiation in human skin in vivo (Chung et al., 2002). In addition, diverse proteinases from the chronic UV induced inflammatory infiltrate can destroy the microfibrils (Kielty et al., 1994).

Therefore, we investigated the effect of ALA-PDT on solar elastosis, and the expression of tropoelastin, fibrillin-1, MMP-12, and inflammatory cells. ALA-PDT reduced the accumulation of dystrophic elastotic material in the dermis, and resulted in more normal elastin fiber. In addition, tropoelastin and fibrillin-1, which were mainly co-localized with the elastotic materials, showed reduced expression after the PDT, and therefore represented an improvement of the solar elastosis. The expression of MMP-12 and inflammatory infiltrates significantly decreased after treatment, which likely was associated with the changes in the solar elastosis as well.

Our findings showed a decrease in the intensity of MMP-1, 3, and 12 immunostaining

following treatment with ALA-PDT. This conflicts with the findings of some other studies that have reported on the effects of ALA-PDT on the expression of MMPs. For example, when normal or scleroderma fibroblast cells were treated with ALA-PDT, the levels of MMP-1 and MMP-3 proteins increased; this was interpreted as antisclerotic effects of ALA-PDT (Karrer et al., 2003). The expressed level of MMPs increased in a time-dependent manner with the maximal induction at 48 hours following the PDT, thereafter with a decreasing tendency.

Recently, in an in vivo study of PDT using a pulsed dye laser in human skin, MMP-1 gene expression was acutely elevated and then returned to baseline levels within 24 hours (Orringer et al., 2008). However, it was also shown that MMP-2 expression was down-regulated 24 hours after Hexvix mediated PDT in a medulloblastoma cell line (TE-671) (Chu et al., 2008).

Our results regarding decreased levels of MMPs might be explained as follows. We evaluated the histological changes one month after the PDT, suggesting that the point in time of the assessment might be an important consideration. In addition, various cell lines used in the in vitro studies might differ from results from our in vivo study. TGF-β might play a role as well.

TGF-β does not only up-regulate procollagen synthesis, but it also down-regulates the

expression of MMPs (Massague, 1990). In the present study, a marked increase of TGF-β expression was noted after the PDT. In addition, a change in the inflammatory infiltrates could affect the decrease in the MMPs. It is known that UV-induced MMPs are secreted by diverse cells, including infiltrated inflammatory cells (Hase et al., 2000). We found that the inflammatory infilates were significantly decreased after the PDT. Therefore, the net effects of the induction of TFG-β and reduction of the degradating enzymes in the inflammatory cells likely contributed to the decreased expression of MMPs after the ALA-PDT (Rijken et al., 2005).

V. CONCLUSION

In conclusion, this study provided histological evidence of the beneficial effects of ALA-PDT for photodamaged skin. In the photodamagend human skin, ALA-PDT induces deposition of collagen, type I and type III procollagen in the dermis and TGF-β and TβR II expression in the

epidermis. In addition, MMP-1 and -3 expression was decreased after ALA-PDT. Solor elastosis was improved accompanying with decreased expression of tropoelastin, fibrillin-1 and MMP-12 as well. These results suggest that ALA-PDT could be of therapeutic benefit in the treatment of photoaging. Further controlled split –face studies on the mechanism of the effects of ALA-PDT on photoaging, including signaling pathways, are necessary in order to fully elucidate the function of ALA-PDT in human skin.

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-국문 요약-

광선 광선

광선 광선 각화증 각화증 각화증 각화증 환자에게서 환자에게서 환자에게서 5-Aminolevulinic Acid 를 환자에게서 를 를 이용한 를 이용한 이용한 이용한 광역동

광역동

광역동 광역동 치료 치료 치료 후 치료 후 후 후 나타난 나타난 나타난 나타난 광회춘 광회춘 광회춘 광회춘 효과 효과 효과 효과: 조직학적 조직학적 조직학적 분석 조직학적 분석 분석 분석

아주대학교 대학원 의학과 박민영

(지도교수: 김유찬)

연구 배경: 만성적인 자외선 노출은 내인성 노화와는 다르고 더 심한 양상을 나타내는 광노화를 유발한다. 조직학적으로 광노화된 피부는 collagen 합성의 감소, 기존 collagen 파괴의 증가와 함께 비정상적인 elastotic material 의 진피 침착을 특징으로 한다. 광노화된 피부를 회복시키기 위한 여러 가지 치료법 중에서 광역동 치료가 이에 효과가 있다는 연구들이 있다. 그러나 이들 대부분의 보고는 임상적인 관찰에 근거한 것이었다.

연구 목적: 본 연구에서는 5-aminolevulinic acid (ALA)를 이용한 광역동 치료가 광 회 춘 효 과 를 나 타 내 는 조 직 학 적 변 화 를 일 으 키 는 지 확 인 하 고 자 하 였 다 .

연구 방법: 얼굴 부위에 한 개에서 세 개의 광선 각화증을 가진 환자를 대상으로, 1200W metal halogen lamp 를 광원으로 하여 한 달 간격으로 2 회 ALA 광역동 치료를 시행하였다. 치료 전과 최종 치료 1 달 후 조직 검사를 시행하였고 총 25 쌍의 피부 조직을 얻었다. 각각에 대하여 기본 염색과 여러 면역 조직 화학 염색을 시행하였다.

문서에서 Photorejuvenation Induced by 5-Aminolevulinic Acid Photodynamic Therapy in the Patients with Actinic Keratosis: A Histological Analysis (페이지 15-39)