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. 2011 Mar;5(1):69-80.
doi: 10.1007/s12079-010-0114-y. Epub 2011 Jan 11.

Spatial-temporal modulation of CCN proteins during wound healing in human skin in vivo

Laure Rittié  1 , Bernard Perbal, John J Castellot Jr, Jeffrey S Orringer, John J Voorhees, Gary J Fisher
Affiliations

Spatial-temporal modulation of CCN proteins during wound healing in human skin in vivo

Laure Rittié et al. J Cell Commun Signal. 2011 Mar.

Abstract

CCN proteins are important modulators of development and function of adult organs. In this study, we examined the localization and expression of the six CCN family members in normal adult human skin and during wound healing in vivo. Transcript and protein expression were studied by laser-capture microdissection-coupled real-time PCR and immunohistochemistry, respectively. Our results demonstrate that CCN1, CCN4, and CCN6 are expressed at relatively low levels in normal human skin. CCN2, CCN3, and CCN5 are the most highly expressed transcripts in the epidermis. CCN3 and CCN5 proteins are prominent in epidermal keratinocytes, whereas CCN2 is primarily expressed in melanocytes. Differential expression within epidermal layers suggests that CCN3 and CCN5 are linked with keratinocyte differentiation. CCN2, CCN3 and CCN5, are the three most highly expressed transcripts in the dermis. Their respective proteins are produced to various extents by dermal fibroblasts, blood vessels, eccrine sweat glands and hair follicles. We find that most CCN family members are temporally and specifically regulated during different phases (inflammation, proliferation, and remodeling) of partial thickness wound repair. By highlighting spatial-temporal regulations of CCN family member expression in relation to cell proliferation and differentiation, our results suggest a diverse range of functions for CCN proteins in both epidermal and dermal cells, and provides a solid reference for interpretation of future studies aimed at understanding the role of CCN proteins in human skin physiology and diseases.

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Figures

Fig. 1
Fig. 1
Distribution and expression levels of CCN transcripts in young adult forearm human skin in vivo. Frozen human forearm skin samples were submitted to laser capture microdissection to separate epidermis from dermis as described in Material and methods. CCN mRNA levels were quantified by qPCR in a epidermis and b dermis. Data are presented relative to housekeeping gene 36B4. N = 9
Fig. 2
Fig. 2
Expression of CCN2, CCN3, and CCN5 proteins in human forearm skin. Skin samples were stained for CCN2 (a–b), CCN3 (c–d), and CCN5 (e–f) by immunohistochemistry. a, c, e: epidermis and papillary dermis; b, d, f deep dermis. Positive staining appears red. Left inset in c indicates papillary dermis blood vessel from a different field. Scale bar, 50 μm
Fig. 3
Fig. 3
Vascular and epidermal changes induced by CO2 laser wounding in human forearm skin. a CO2 laser creates a partial thickness wound by vaporizing the entire epidermis and the upper part of the papillary dermis. Basement membrane is highlighted by lamin-γ2 staining for reference. Scale bar, 100 μm. b Time course of blood vessel formation after wounding in human skin. Endothelial cells are stained with CD31. Blood vessel formation starts within the first week post-wounding, and hyper-vascularization is visible for at least 4 weeks. Scale bar, 100 μm. c Time course of re-epithelialization of partial thickness wounds in human skin. Proliferative cells are stained with Ki67. Epidermal hyperplasia is visible at weeks 2 and 3, and epidermal thickness is normalized by week 4 (also visible on b). Scale bar, 100 μm
Fig. 4
Fig. 4
CCN mRNA expression in the epidermis following CO2 laser wounding in human forearm skin. Skin samples were obtained from non-treated areas and 7, 14, 21, and 28 days post-CO2 laser treatment. Frozen skin sections were submitted to laser capture microdissection to isolate epidermis as described in Material and methods. CCN mRNA were quantified by qPCR in epidermis samples: a CCN1, CCN2, CCN4, CCN6. Differences were not statistically different to “no treatment” at all time-points; b CCN3; c CCN5. Data are relative to housekeeping gene 36B4. N = 3–5; *: P < 0.05 vs. no treatment; NS not significant vs. no treatment
Fig. 5
Fig. 5
CCN3 and CCN5 protein expression in epidermis and papillary dermis following CO2 laser wounding in human forearm skin. Skin samples were obtained from non-treated area and 14, 21, and 28 days post-CO2 laser treatment, and were analyzed for a CCN3 and b CCN5 proteins by immunohistochemistry. In panel A, note the nuclear CCN3 protein in the upper epidermal layers at weeks 2 and 3 (black arrow), in sharp contrast with the absence of CCN3 protein in the same upper layers at week 4 (white arrow). a,b Representative of three subjects. Scale bars, 100 μm
Fig. 6
Fig. 6
CCN mRNA expression in the dermis following CO2 laser wounding in human forearm skin. Skin samples were obtained from non-treated area and 1, 2, 3, 7, 14, 21, and 28 days post-CO2 laser treatment. Frozen skin sections were submitted to laser capture microdissection to isolate dermis as described in Material and methods. CCN transcripts were quantified by qPCR. Time course of expression of CCN mRNA in dermis: a CCN1; b CCN2; c CCN3; d CCN5. Data are relative to housekeeping gene 36B4. N = 3–5; *: P < 0.05 vs. no treatment; NS: not significant vs. no treatment. e Ratio of CCN2 to CCN5 from data presented in b and d; CCN2 and CCN5 levels are expressed as % of (CCN2 + CCN5) mRNA levels. N = 3–5
Fig. 7
Fig. 7
CCN2 protein expression during dermal repair. Skin samples were obtained from non-treated area and 7, 21, and 28 days post-CO2 laser treatment, and analyzed for CCN2 protein by immunohistochemistry. Representative of 3 subjects. Scale bars, 50 μm
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