SKINIPEDIA - Skin Diseases: Reverse Skin Atrophy Results
Kim Yancey, Academic Editor
Medical College of Wisconsin, United States of America
* To whom correspondence re: this article should be addressed. E-mail: gkaya@hcuge.ch
DISCUSSION
Our observations provide evidence for the first time to our knowledge that topically applied HAF ranging from 50,000 to 400,000 Da penetrate the epidermis and induce keratinocyte proliferation that translates into the thickening of mouse and human skin. Penetration of topically applied HA of 250,000–400,000 Da into mouse and human dermis was recently demonstrated [21]. Here we show that both HAFs and HAFi penetrate mouse skin in vivo, but that only the HAFi induce cellular proliferation within the epidermal and dermal compartments.
At least two types of effects occurred as a result of topical HAFi application: CD44-independent penetration of skin and induction of HAS and Hyal2, and CD44-dependent keratinocyte proliferation. Both fibroblasts and endothelial cells also proliferated in response to HAFi in vitro, providing a possible explanation for the increased dermal collagen deposition and angiogenesis, respectively, observed in vivo. However, the contribution of fibroblast activity and angiogenesis to epidermal hyperplasia was most likely of minor importance, given that conditioned culture media of HAFi-stimulated fibroblasts and endothelial cells failed to augment keratinocyte proliferation. Importantly, an increase in human skin thickness in response to HAFi was observed only in patients with skin atrophy. Although the reasons of the absence of such a response in healthy participants can only be speculative at present, it is possible that physiological HA production saturates tissue CD44 binding capacity in the steady state, such that additional exogenous HA fragments fail to induce a significant CD44-dependent response. Whatever the precise mechanism, the absence of skin hyperplasia in healthy participants in response to HAFi suggests that topical HAFi administration does not present the risk of inducing undesirable local side effects.
Penetration of HA fragments into the dermis may occur via hair follicles, which provide a well-recognized route for macromolecular skin penetration [22] and could explain the dermal localization of HAFs and HAFi. However, the increase in dermal HA content of HAFi- and HAFs-treated hairless SKH1 mouse skin, which contains incompletely developed and partially functional follicles [22], leaves open the possibility for the participation of an additional putative mechanism of size-limited HAF penetration, including possibly passive absorption. HAFi was observed to induce HAS expression consistent with the possibility that increased local HA synthesis may contribute to the increased HA content. The mechanism whereby HAFi might induce local HAS and hyaluronidase expression is unknown. Keratinocytes and fibroblasts provide the principal source of HA in the epidermis and dermis, respectively [23], and control local HA metabolism. CD44 is believed to play a major role in the uptake of HA by receptor-mediated endocytosis [24] in both cell types. Following internalization, HA undergoes intracellular degradation that is possibly mediated by endosomal/lysosomal hyaluronidases [25]. HAF released by keratinocytes traverse the basement membrane to the dermis, where they are cleared via lymphatic vessels [26]. It would appear that mechanisms independent of CD44 might sense changes in local HA concentration, or that HAFi stimulate receptors other than CD44 to induce HAS and hyaluronidase expression. Increased tissue HA concentration typically occurs in the context of development, injury, and tumor growth, and is believed to actively participate in the process of tissue remodeling. It is conceivable that an increase in tissue HA, whether of endogenous or exogenous origin, is interpreted by local fibroblasts to reflect a remodeling process, triggering additional HA synthesis and degradation.
Proliferation in response to HAFi is a CD44-dependent event. Our present observations provide evidence that in addition to CD44, HB-EGF, erbB1, and MMPs/ADAMs are required for HA-dependent in vitro keratinocyte proliferation. The absence of a proliferative response of CD44−/− keratinocytes to HB-EGF is consistent with the notion that HB-EGF interaction with its receptors requires presentation by heparan sulfate side chains of CD44v3-containing isoforms. Similar to other members of the EGF family, pro-HB-EGF is expressed as an integral membrane protein of 22–28 kDa, which, following stimulation of cells with mitogens, undergoes MMP-mediated release of mature soluble 14–19 kDa HB-EGF. Accordingly, topical application of HAFi oligosaccharide resulted in a significant increase in pro-HB-EGF and HB-EGF, while inhibition of MMP activity by TIMP-3 abrogated the corresponding proliferative response.
The observation that anti-erbB1– and anti–HB-EGF–blocking antibodies had the same abrogating effect as the absence of CD44 on in vitro keratinocyte proliferation in response to HAFi supports the notion that erbB1 signaling, triggered by HB-EGF, may play a key role in HAFi-induced proliferation. Similar to its role in uterine and mammary epithelia, HA-induced CD44v3 aggregates may recruit a functional cell-surface complex in keratinocytes composed of pro-HB-EGF, erbB1, which replaces erbB4, and an MMP or ADAM [13,20]. MMP/ADAM-mediated proteolytic cleavage of HB-EGF and CD44-mediated presentation of mature HB-EGF to erbB1 may then induce proliferation (Figure 5).
Editorial Summary to Reverse Skin Atrophy