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Differential Effects of Photoaging vs Intrinsic Aging on the Vascularization of Human Skin
Jin Ho Chung, MD;
Kiichiro Yano, PhD;
Mi Kyung Lee, MD;
Choon Shik Youn, MD;
Jin Young Seo, MD;
Kyu Han Kim, MD;
Kwang Hyun Cho, MD;
Hee Chul Eun, MD;
Michael Detmar, MD
Arch Dermatol. 2002;138:1437-1442.
ABSTRACT
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Objective To quantify the distinct effects of photoaging vs intrinsic aging of human skin on cutaneous vascularization in the Korean population.
Design Case series.
Setting University hospital.
Participants A total of 21 healthy Korean volunteers from the third to the ninth decades of life.
Intervention Skin biopsy specimens were obtained from chronically sun-exposed and sun-protected skin of each participant.
Main Outcome Measures Frozen sections were stained for the platelet endothelial cell adhesion molecule CD31 (PECAM-1), and computer-assisted quantitative image analysis was performed to quantify cutaneous vascular density and vessel size.
Results Intrinsically aged and photoaged skin showed an age-dependent reduction of cutaneous vessel size. However, only photoaged skin exhibited significantly reduced numbers of dermal vessels, in particular in the subepidermal areas that displayed extensive matrix damage. Linear regression analysis revealed an inverse relation of vessel numbers and age in sun-damaged, but not in sun-protected, skin.
Conclusions In Korean skin, chronic photodamage results in a gradual decrease in the number and size of dermal vessels over several decades of sun exposure, most likely due to degenerative changes of the dermal extracellular matrix. Because the present investigation was restricted to ethnic Korean volunteers, future studies are needed to evaluate whether similar changes can be observed in whites.
INTRODUCTION
PHOTOAGING OF human skin is caused by repeated exposures to UV radiation over a prolonged period. In contrast, intrinsic skin aging is related to naturally occurring biological aging processes in sun-protected skin. Intrinsically aged skin appears smooth, pale, and finely wrinkled. In contrast, photoaged skin is coarsely wrinkled and is frequently characterized by abnormal pigmentation and telangiectasias. The most dramatic histologic differences between intrinsically aged and photoaged skin occur within the dermis and involve degradation of a number of extracellular matrix proteins, including collagen and elastic fibers.1-3 A reduction of the cutaneous microvasculature has been observed in the skin of older individuals,4-5 potentially leading to reduced nutritional support of older skin.6 Moreover, obliterated vessels have been associated with disturbances of the normal architecture of vascular plexus in the dermis.4 In contrast, no major disturbances of the horizontal pattern of vascular plexus have been found in intrinsically aged skin.7
Previous studies of the cutaneous vasculature in intrinsically aged and photoaged skin have almost exclusively focused on end-stage dermal changes, whereas a detailed study of the sequence of vascular alterations occurring over decades has been lacking. In particular, the sequential vascular changes occurring during distinct stages of photoaging have remained unclear. Moreover, a quantitative, morphometric analysis of the distinct changes of dermal microvessel size and numbers during intrinsic skin aging vs cutaneous photoaging has been lacking. In the present study, we performed a computer-assisted, quantitative image analysis of the vascular changes that occur during intrinsic skin aging and throughout the successive steps of photoaging in Koreans, with a particular focus on the sequential alterations of vessel size and density over several decades.
METHODS
BIOPSY SPECIMENS
Two- and 4-mm punch biopsy specimens were obtained from chronically sun-exposed skin (face; crow's feet area) and sun-protected skin (buttock), respectively (Figure 1), of 21 adult Koreans (12 men, 9 women) without current or prior skin disease.8 Three volunteers were included for each decade of life from the third to the ninth. All human studies were approved by the institutional review board of the Seoul National University Hospital, and all subjects gave written informed consent.
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Figure 1. Representative photographs of sun-exposed skin in a 25-year-old subject (A) and a 77-year-old subject (B). C, Chronically sun-exposed, left, and sun-protected, right, skin of a 76-year-old individual.
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IMMUNOHISTOCHEMICAL ANALYSIS FOR CD31 AND COMPUTER-ASSISTED MORPHOMETRIC ANALYSIS OF CUTANEOUS BLOOD VESSELS
Specimens were oriented immediately into a cryomatrix (Shandon, Pittsburgh, Pa), snap-frozen in liquid nitrogen, and then stored at -70°C. Frozen sections 8 µm thick were mounted onto silane-coated slides (Dako, Glostrup, Denmark) and acetone fixed at -20°C for 15 minutes. Thereafter, sections were incubated with a mouse 1:200 monoclonal antihuman CD31 antibody (Pharmingen, San Diego, Calif) for 1 hour at room temperature. After being rinsed in phosphate-buffered saline, the sections were incubated with a biotinylated secondary antimouse IgG antibody and horseradish-streptavidin conjugate, using the LSAB kit (Dako). The chromogenic substrate used was 3-amino-9-ethylcarbazole. Sections were counterstained in Mayer hematoxylin. Control stainings were performed with the same amount of normal mouse immunoglobulin (IgG1) and showed no immunoreactivity (data not shown).
Representative sections were analyzed using a Nikon E-600 microscope (Nikon, Melville, NY), and images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, Mich). Morphometric image analyses were performed using the IP-LAB software (Scanalytics Inc, Fairfax, Va) as previously described.9 Three different fields of each section were examined at x60 magnification, and the number of vessels per square millimeter, the average vessel size, and the relative area occupied by blood vessels were determined in the dermis, in an area within 500 µm of the dermoepidermal junction.10
Computer-assisted morphometric analysis of CD31-stained sections was performed in a blinded way to avoid bias. The 2-sided unpaired t test was used to analyze differences in microvessel density and vascular size. Linear regression analyses were performed using StatView software version 4.5 (SAS Institute Inc, Cary, NC).
RESULTS
AGE-DEPENDENT REDUCTION OF CUTANEOUS VASCULARITY IN CHRONICALLY SUN-DAMAGED KOREAN SKIN
Histologic analysis of skin samples obtained from chronically sun-exposed skin of healthy volunteers confirmed progressive thinning of the epidermis, reduction of epidermal rete ridge formation, and dermal photodamage manifested by elastosis in older individuals compared with younger individuals (Figure 2A, C, and E). To investigate the effects of chronic photodamage on cutaneous vascularity, we performed immunostains with a specific antibody for the CD31 antigen (platelet endothelial cell adhesion molecule [PECAM-1]), an endothelial junction molecule. We found that chronic photodamage was associated with dramatically reduced numbers of cutaneous vessels that were also reduced in size (Figure 2B, D, and F).
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Figure 2. Increased extent of cutaneous photodamage is associated with markedly reduced cutaneous vascularity. Representative skin samples obtained from chronically sun-exposed skin of 3 healthy subjects (ages given at right) demonstrate increasing dermal photodamage, epidermal thinning, and reduced rete ridge formation (A, C, and E; hematoxylin-eosin [H&E], original magnification x10) associated with progressively diminishing numbers of cutaneous vessels (B, D, and F; immunostains for the endothelial junction molecule CD31, original magnification x10).
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To quantify the changes of skin vascularization during photoaging, we next performed computer-assisted morphometric analyses of CD31-stained skin sections. We compared vessel numbers, average vessel size, and the percentage of cutaneous area covered by vessels in 3 groups of individuals: (1) aged 20 to 39 years (n = 6); (2) aged 40 to 69 years (n = 9); and (3) aged 70 to 84 years (n = 6). These studies revealed that vessel numbers were significantly reduced in the photodamaged skin of individuals 70 years and older (-43.1%; P<.001) (Figure 3A). Moreover, significant decreases in average vessel size were already detected in individuals aged 40 to 69 years (-31.8%; P<.01) with an even more pronounced reduction after age 70 years (-45.3%; P = .001) (Figure 3B). In accordance with these findings, the average dermal area covered by vessels, the most sensitive parameter for measuring skin vascularity, was reduced by 43% in subjects aged 40 to 69 years (P = .02) and by 69.5% in subjects 70 years and older (P<.001) (Figure 3C).
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Figure 3. Reduced vessel numbers and size in photoaged skin. Computer-assisted image analysis of CD31-stained sections obtained from chronically sun-exposed skin demonstrates progressively diminishing vessel density (A) and vessel size (B), resulting in a significantly reduced dermal area covered by CD31-stained vessels (C). Three groups of volunteers were compared: (1) aged 20 to 39 years (n = 6); (2) aged 40 to 69 years (n = 9); and (3) aged 70 to 84 years (n = 6). Error bars indicate SDs; P values are as compared with group 1, and where no P value is indicated, the difference was not significant.
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Linear regression analysis of the individual age and the results of the computer-assisted morphometric analysis of vascular parameters demonstrated a strong negative correlation of age with vessel density (r = 0.830) (Figure 4A), vessel size (r = 0.814) (Figure 4B), and dermal area covered by CD31-stained vessels (r = 0.879) (Figure 4C), confirming the progressive development of vascular regression with increasing photodamage of the skin.
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Figure 4. Inverse correlation between age and dermal vascularity in chronically sun-exposed skin. Linear regression analysis of the donor age with the computer-assisted morphometric analysis of vascular parameters demonstrates a strong negative correlation of age with vessel density (A) (r= 0.830), vessel size (B) (r= 0.814), and dermal area covered by CD31-stained vessels (C) (r= 0.879).
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DECREASED VESSEL SIZE IN SUN-PROTECTED SKIN OF OLDER KOREAN SUBJECTS
Histologic analyses of sun-protected skin did not reveal any major morphologic changes except a slight thinning of the epidermis and modest flattening of the dermoepidermal junction in older individuals (Figure 5A, C, and E). In contrast to photodamaged skin, increased age was not associated with reduced vessel numbers in sun-protected skin (Figure 5B, D, and F), as demonstrated by immunostains for CD31. However, with increasing age, the size of dermal vessels appeared to be smaller in older skin (Figure 5D and F) than in the skin of younger individuals (Figure 5B).
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Figure 5. Increased age is not associated with reduced vessel numbers in sun-protected skin. Representative skin samples obtained from sun-protected skin of 3 healthy subjects (ages given at right) demonstrate moderate thinning of the epidermis (A, C, and E; hematoxylin-eosin [H&E], original magnification x10) but no reduction of vessel numbers in the dermis (B, D, and F; immunostains for the endothelial junction molecule CD31, original magnification x10). However, with increasing age, the size of dermal vessels is reduced.
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Image analysis of CD31-stained sections confirmed that no major reduction of vessel numbers occurred during intrinsic skin aging (+8.5% in individuals aged 40-69 years [not significant]; -2.1% in individuals  70 years [not significant]) Figure 6A). In contrast, we detected a significant decrease in average vessel size (-30.6% in individuals aged 40-69 years [P = .01] and -30.8% in individuals 70 years [P = .01]) (Figure 6B). Overall, the cutaneous area covered by CD31-positive vessels was only moderately changed, and the differences among the 3 age groups did not reach statistical significance (-29.7% for subjects aged 40-69 years [not significant] and -26.4% for subjects 70 years [not significant]) (Figure 6C). In accordance with these results, linear regression analysis of the individual age and the results of the vascular parameters did not detect a significant correlation between age and vessel density (r = 0.068) (Figure 7A), vessel size (r = 0.543) (Figure 7B), or cutaneous area covered by vessels (r = 0.333) (Figure 7C).
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Figure 6. Unchanged vessel numbers and moderately reduced vessel size in intrinsically aged skin. Computer-assisted image analysis of CD31-stained sections obtained from sun-protected skin demonstrates comparable vessel densities (A) and moderately reduced average vessel size (B), resulting in a minor reduction of the dermal area covered by CD31-stained vessels (C). Three groups of volunteers were compared: (1) aged 20 to 39 years (n = 6); (2) aged 40 to 69 years (n = 9); and (3) aged 70 to 84 years (n = 6). Error bars indicate SDs; P values are as compared with group 1.
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Figure 7. Lack of correlation between age and dermal vascularity in intrinsically aged skin. Linear regression analysis of the donor age with the computer-assisted morphometric analysis of vascular parameters revealed absence of correlation of age with vessel density (A) (r= 0.068) and no significant correlation between age and vessel size (B) (r= 0.543) or dermal area covered by CD31-stained vessels (C) (r= 0.333).
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COMMENT
In the present study, we have quantified the sequential changes of cutaneous vascularity during photoaging of chronically sun-exposed human skin and during the intrinsic aging of sun-protected skin in Koreans over a period of 7 decades. Using computer-assisted image analysis of tissue sections stained for the endothelial cell junction molecule CD31 (PECAM-1), we investigated the changes in vessel numbers and size during intrinsic aging and during photoaging. Our results reveal that photoaged Korean skin shows a progressive loss of dermal blood vessels associated with a significant reduction in the average vessel size, predominantly in the upper dermis. In contrast, no major changes of cutaneous vessel density were detected in intrinsically aged photoprotected skin, even in a group of individuals aged 70 to 89 years. However, intrinsically aged skin was characterized by a significant reduction of the average vessel size. Previous studies have mainly focused on vascular changes and reduced vascularity associated with high-grade chronic photodamage or with chronologically aged skin.3, 6-7,11 A reduction in venular cross sections has been shown in the papillary dermis of buttock skin from elderly adults compared with skin obtained from young adult controls,7 and a significant reduction of dermal papillary loops has been detected in photoaged skin obtained from the forehead and forearm compared with young skin.6
Our finding that vessel numbers were dramatically reduced in photodamaged skin but not in aged sun-protected skin strongly suggests that different molecular mechanisms are involved in the mediation of the vascular alterations in both conditions. Acute UV-B irradiation results in marked up-regulation of proangiogenic and proinflammatory mediators, including vascular endothelial growth factor, with resulting vascular hyperpermeability, vessel leakage, activation of proteases, and degradation of extracellular matrix molecules. These changes are associated with vascular proliferation and upregulation of endothelial cell adhesion molecules with consecutive influx of inflammatory cells into the dermis. Taken together, these changes represent an inflammatory tissue repair reaction and, after chronic and repetitive damage, result in marked degenerative changes of the upper dermis with degradation of elastic and collagen fibers and rarefication of cutaneous vessels, likely providing a less permissive environment for the maintenance of normal vessel structure and function. One has to keep in mind, however, that the present results were exclusively obtained in aged skin type V obtained from Korean individuals. Future quantitative studies are needed to specifically quantify the sequential changes of cutaneous vascularity in whites with skin types I through IV who frequently develop increased redness of facial skin with increasing age.
In sun-protected skin, no major tissue repair/damage reactions or vascular activation occur over a period of several decades, and only minor changes of the extracellular matrix are detected in intrinsically aged skin. Accordingly, we did not detect a major reduction in the number of vessels in intrinsically aged human skin, whereas the average size of cutaneous vessels was significantly reduced. The age-associated decrease of dermal vessel size may explain several of the physiologic alterations characteristic of aged skin, including pallor, decreased skin temperature, reduced cutaneous vascular responsiveness, and reduced UV-induced erythema.4-5,7 The mechanisms leading to the observed reduction in average vessel size remain unknown. Because vascular endothelial cells are normally characterized by low levels of cell proliferation and because most cutaneous vessels are usually quiescent, it appears rather unlikely that endothelial cells in aged sun-protected skin might have reached the stage of replicative senescence. It remains to be investigated whether aged skin is characterized by reduced expression or receptor signaling12 of distinct angiogenesis or vascular remodeling factors, including several members of the vascular endothelial growth factor family, thrombospondins, and angiopoietins that have been previously shown to modulate blood vessel size.13
It has been reported that topical treatment of human skin with retinoic acid14-15 may stimulate cutaneous angiogenesis. Long-term treatment of human skin with topical retinoic acid has resulted in increased dermal vascularity and in the reappearance of superficial capillary loops, often in close apposition to the epidermis.16-17 Our results suggest that the beneficial effects of retinoic acid on skin vascularity might constitute a major component of its mechanism of action in chronically photodamaged skin, leading to restoration of blood flow to the subepidermal areas of the dermis that display the most prominent loss of vascularization after chronic sun exposure. In fact, several of the clinical and histologic skin changes observed after tretinoin treatment, including epidermal thickening, require increased blood flow that can accommodate the increased nutritional needs of the epidermis and dermis.17-18 It remains to be established whether the effects of retinoic acid on the vascularity of aged skin are due to direct stimulation of blood vessel growth or the restoration of a more normal dermal extracellular matrix, providing a more permissive environment for vessel growth and maintenance.
The present investigation provides the rationale for future studies into the potential beneficial effects of specific angiogenic mediators for the treatment of late-stage photodamaged skin in older individuals. However, recent results suggest that proangiogenic mediators are involved in the mediation of acute and subacute UV-B–induced skin damage19-20 and that absence of normally present endogenous angiogenesis inhibitors results in enhanced dermal photodamage (K. Yano et al, unpublished data, 2002). Moreover, blockade of skin angiogenesis by targeted overexpression of the natural angiogenesis inhibitor thrombospondin-1 in epidermal keratinocytes largely prevented UV-B–induced cutaneous damage and wrinkle formation in mice,19 indicating that UV-B–induced cutaneous angiogenesis and vascular activation contribute to the resulting dermal damage and that antiangiogenic strategies might prevent the development of cutaneous photodamage. Taken together, these findings provide increasing evidence for a crucial role of cutaneous blood vessels in the pathogenesis of photoaging of the skin, and different mechanisms are likely involved in the vascular mediation of acute photodamage vs the chronic hypovascularity of older sun-exposed skin. Elucidation of the molecular pathways that are responsible for the pronounced vascular abnormalities observed in these conditions may provide new targets for the development of therapies aimed at the prevention or treatment of cutaneous photodamage.
AUTHOR INFORMATION
Accepted for publication July 24, 2002.
This work was supported by grants CA69184 and CA86410 from the National Institutes of Health/National Cancer Institute, Bethesda, Md (Dr Detmar); a Life Phenomena and Function Research Group Program grant from the Korean Ministry of Science and Technology, Seoul, Korea (Dr Chung); a research agreement with Pacific Corporation, Seoul (Dr Chung); and by the Cutaneous Biology Research Center, Charlestown, Mass, through the Massachusetts General Hospital/Shiseido Co Ltd Agreement (Dr Detmar).
Corresponding author: Jin Ho Chung, MD, Department of Dermatology, Seoul National University Hospital, 28 Yungon-dong, Chongno-Gu, Seoul 110-744, Korea (e-mail: jhchung{at}snu.ac.kr).
From the Department of Dermatology, Seoul National University College of Medicine, and Laboratory of Cutaneous Aging Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea (Drs Chung, Lee, Youn, Seo, Kim, Cho, and Eun); The Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston (Drs Yano and Detmar).
REFERENCES
| |
1. Gilchrest BA. Skin aging and photoaging: an overview. J Am Acad Dermatol. 1989;21:610-613.
ISI
| PUBMED
2. Lavker RM. Structural alterations in exposed and unexposed aged skin. J Invest Dermatol. 1979;73:559-566.
3. Lavker RM, Kligman AM. Chronic heliodermatitis: a morphologic evaluation of chronic actinic dermal damage with emphasis on the role of mast cells. J Invest Dermatol. 1988;90:325-330.
FULL TEXT
|
ISI
| PUBMED
4. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol. 1979;73:39-46.
FULL TEXT
|
ISI
| PUBMED
5. Yaar M, Gilchrest BA. Aging of skin. In: Freedberg IM, Eisen AZ, Wolff K, et al, eds. Dermatology in General Medicine. 5th ed. New York, NY: McGraw-Hill; 1999:1697-1706.
6. Kelly RI, Bull RH, Leveque JL, Rigal J, Mortimer PS. The effects of aging on the cutaneous microvasculature. J Am Acad Dermatol. 1995;33:749-756.
FULL TEXT
|
ISI
| PUBMED
7. Gilchrest BA, Stoff JS, Soter NA. Chronologic aging alters the response to ultraviolet-induced inflammation in human skin. J Invest Dermatol. 1982;79:11-15.
FULL TEXT
|
ISI
| PUBMED
8. Chung JH, Lee SH, Youn CS, et al. Cutaneous photodamage in Koreans: influence of sex, sun-exposure, smoking, and skin color. Arch Dermatol. 2001;137:1043-1051.
FREE FULL TEXT
9. Streit M, Velasco P, Riccardi L, et al. Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice. EMBO J. 2000;19:3272-3282.
FULL TEXT
|
ISI
| PUBMED
10. Yano K, Brown LF, Detmar M. Control of hair growth and follicle size by VEGF-mediated angiogenesis. J Clin Invest. 2001;107:409-417.
ISI
| PUBMED
11. Herzberg AJ, Dinehart SM. Chronologic aging in black skin. Am J Dermatopathol. 1989;11:319-328.
ISI
| PUBMED
12. Yaar M, Gilchrest B. Aging versus photoaging: postulated mechanisms and effectors. J Investig Dermatol Symp Proc. 1998;3:47-51.
PUBMED
13. Gale NW, Yancopoulos G. Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins and ephrins in vascular development. Genes Dev. 1999;13:1055-1066.
FREE FULL TEXT
14. Kligman AM, Grove GL, Hirose R, Leyden JJ. Topical tretinoin for photoaged skin. J Am Acad Dermatol. 1986;15:836-859.
ISI
| PUBMED
15. Bhawan J. Short-and long-term histologic effects of topical tretinoin on photodamaged skin. Int J Dermatol. 1998;37:286-292.
FULL TEXT
|
ISI
| PUBMED
16. Kligman AM, Dogadkina D, Lavker RM. Effects of topical tretinoin on non-sun-exposed protected skin of the elderly. J Am Acad Dermatol. 1993;29:25-33.
ISI
| PUBMED
17. Griffiths CEM, Kang S, Ellis CN, et al. Two concentrations of topical tretinoin (retinoic acid) cause similar improvement of photoaging but different degrees of irritation. Arch Dermatol. 1995;131:1037-1044.
FREE FULL TEXT
18. Detmar M. Molecular regulation of angiogenesis in the skin. J Invest Dermatol. 1996;106:207-208.
FULL TEXT
|
ISI
| PUBMED
19. Yano K, Oura H, Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photodamage. J Invest Dermatol. 2002;118:800-805.
FULL TEXT
|
ISI
| PUBMED
20. Brauchle M, Funk JO, Kind P, Werner S. Ultraviolet B and H2O2 are potent inducers of vascular endothelial growth factor expression in cultured keratinocytes. J Biol Chem. 1996;271:21793-21797.
FREE FULL TEXT
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