Natural and Sun-Induced Aging of Human Skin

Aging of Skin Innervation

Skin connects to the central nervous system through dense innervation. Skin innervation is traditionally described according to the function it mediates, such as providing the senses of touch, itch, or pain (afferent sensory innervation), as opposed to glandular activity or smooth muscle contraction (efferent autonomic innervation) (Montagna 1977; Fraiture et al. 1998; Reinisch and Tschachler 2012). Aging is associated with an overall decreased sensory perception that correlates with reduced number of nerve fiber endings in epidermis and dermis (Grassi et al. 2003; Panoutsopoulou et al. 2009; Fromy et al. 2010; Namer 2010). On the other hand, a few studies have shown that photoaged skin is characterized by increased sensory nerves (Toyoda et al. 2005) and increased number of epidermal nerve fibers that correlates with the intensity of photoaging (Toyoda et al. 1996) compared with sun protected skin. The functional consequences of these changes in photoaged skin remain hypothetical (Legat and Wolf 2006, 2009).

Aging of Skin Immunity

Skin’s natural barrier is aided by a powerful intrinsic immune system to help protect against infection. Antigen-presenting cells, which traffic throughout the skin, include macrophages, B cells, dendritic cells, and Langerhans cells (the latter being the major antigen-presenting cell in the epidermis). Recent reviews have covered, in detail, age-associated changes in the skin immune systems (Ongrádi et al. 2009; Shaw et al. 2010; Mahbub et al. 2011; Vukmanovic-Stejic et al. 2011). In general, aging is associated with deregulation of the immune response, often referred to as “immunosenescence,” which translates to increased susceptibility to infections, increased malignancies, and reduced effectiveness of vaccination. Typically, the number of cutaneous antigen-presenting cells is similar between young and aged subjects, but migration to lymph nodes, phagocytosis, and capacity to stimulate T cells is reduced in elderly versus young individuals (Takahashi et al. 1985; Bhushan et al. 2004; Agrawal et al. 2007; Ongrádi et al. 2009; Shaw et al. 2010; Mahbub et al. 2011; Vukmanovic-Stejic et al. 2011). Although acute UV irradiation alters immune response in human skin (Baadsgaard et al. 1989; Bennett et al. 2008), the permanent effects of chronic UV exposure on immunity in photoaged skin remain unclear.

Aging of Skin Vasculature

Skin vasculature is made of a network of blood and lymphatic vessels that resides in the dermis. Skin vasculature allows temperature control and diffusion of nutrient-rich plasma and immune cells. While blood vessels serve as supply lines, lymphatic vessels ensure recycling through back transport to the venous circulation (Armulik et al. 2005; Cueni and Detmar 2006). Blood vessels are formed on interaction between endothelial cells, mural cells (pericytes and vascular smooth muscle cells), and the surrounding extracellular matrix (Armulik et al. 2005; Betsholtz et al. 2005). Aging and photoaging of the skin are associated with reduced number of blood vessels, especially in the upper dermis (Kligman 1979; Braverman and Fonferko 1982; Kelly et al. 1995; Chung et al. 2002). Structural changes in vessel properties also include reduced number of pericyte-covered blood vessels in aged skin (Betsholtz et al. 2005; Helmbold et al. 2006). In addition, endothelial cell flattening and dilation of the remaining vessels are typical of photoaged skin (Kligman 1979). Disruption of blood vessels’ normal architecture leads to vascular hyperpermeability and, thus, dysfunction (Armulik et al. 2005; Betsholtz et al. 2005). For instance, maximum cutaneous blood flow, a measure of skin endothelial function in response to heating, is reduced linearly with age (Martin et al. 1995). Similarly, reduced density of lymphatic vessels has been observed in photoaged human skin (Kajiya et al. 2007). Taken together, changes in vasculature alter multiple aspects of cutaneous physiology as diverse as increased tendency to bruising, reduced nutrient supply, and altered immune cell trafficking.

Acute UV Irradiation Triggers Transient Collagen Fibril Degradation

During the past two decades, we have greatly enhanced our understanding of the mechanisms by which repeated skin exposure to solar irradiation over decades leads to sustained structural and functional deficits in the dermis. Oxidative stress plays a central role in both aging-associated and UV irradiation-initiated dermal extracellular matrix alterations. On irradiating the skin surface, UV light energy is readily absorbed by endogenous cellular chromophores such as NADH⁻/NADPH, tryptophan, riboflavin, or trans-urocanic acid (Hanson and Simon 1998). Chromophore excitation, which occurs in the presence of molecular oxygen, produces an array of oxidation products and radical oxygen species (ROS), including the highly reactive hydroxyl radical HO^• (Brenneisen et al. 1998). Although toxic at high concentration, ROS mediate a variety of normal cellular responses at low concentration, such as activation of receptor tyrosine kinases (RTKs) and downstream signaling pathways (Herrlich and Bohmer 2000). ROS produced upon UV irradiation, likewise, activate these RTKs and their associated downstream signaling pathways, bypassing the normal initiation process of binding cognate ligands. Specifically, ROS reversibly reacts with highly conserved cysteine in the catalytic site and thereby inhibits enzymatic activity of receptor protein tyrosine phosphatases (RPTPs) (Knebel et al. 1996; Denu and Tanner 1998; Xu et al. 2002; Meng and Zhang 2013). These RPTPs directly dephosphorylate RTKs and thereby maintain RTKs in low-activation state levels () (Tonks 2006). Thus, RPTP inhibition is functionally similar to RTK activation. In human keratinocytes, RPTP-κ specifically regulates epidermal growth factor receptor (EGFR) tyrosine phosphorylation (Xu et al. 2005) and mediates UV irradiation-activation of EGFR (Xu et al. 2006). Other protein tyrosine phosphatases are also likely to be affected because UV irradiation activates many other RTKs, including cytokine and growth factor cell surface receptors such as tumor necrosis factor-α receptor, platelet activating factor receptor, insulin receptor, interleukin-1 receptor, CD95 (Fas-Ligand receptor), insulin receptor, and platelet-derived growth factor receptor (Herrlich and Bohmer 2000; Rittié and Fisher 2002). The pleiotropic response induced by UV irradiation in skin derives in large part from the ability of UV light to simultaneously activate multiple RTKs (Rosette and Karin 1996).

ROS-mediated activation of RTKs signaling cascades by UV irradiation. (Upper panel) In absence of UV irradiation (basal conditions), RTKs and downstream signaling in skin cells are maintained in a low state of activation by protein tyrosine phosphatase activities that dynamically dephosphorylate RTKs. These conditions favor normal collagen synthesis and low production of matrix metalloproteinases (MMPs). (Lower panel) Absorption of UV irradiation energy by skin cell components, in the presence of molecular oxygen, generates reactive oxygen species (ROS) that react with cysteine in the catalytic site of protein tyrosine phosphatases. Inhibition of protein tyrosine phosphatases by reaction with ROS increases net RTK phosphorylation levels and triggers downstream signaling cascades that include mitogen-activated protein kinase (MAPK) phosphorylation and activation of activator protein-1 (AP-1) transcription factor. Activated AP-1 represses collagen production and increases MMP gene transcription. As a result, UV irradiation induces transient collagen deficit.

The downstream signaling that follows UV irradiation-mediated RTK activation is similar to that triggered by ligand-binding and has been reviewed in detail elsewhere (Rittié and Fisher 2002). Briefly, downstream signaling consists of recruitment of adaptor proteins and activation of the three families of MAPKs: extracellular signal-regulated kinase, p38, and c-Jun amino-terminal kinase. In human skin, UV irradiation activates MAPKs in the epidermis and upper dermis (Fisher et al. 2002). Activated MAPKs, in turn, phosphorylate the transcription factor c-Jun. c-Jun activation occurs within 30–60 min following UV irradiation of skin in vivo and lasts 24 h (Fisher and Voorhees 1998). Activated c-Jun enters the nucleus and partners with constitutively expressed c-Fos to assemble the activated AP-1 transcription factor complex. AP-1 has a profound influence on collagen homeostasis because it not only stimulates transcription of several collagen- degrading enzymes including MMP-1, -3, and -9, but also reduces production of procollagens (soluble precursors to collagens) by inhibiting transcription of genes encoding procollagens I and III.

Other mechanisms are implicated in mediating altered regulation of extracellular matrix protein-encoding genes by UV irradiation. Among them, deregulation of transforming growth factor β (TGF-β) pathway is of particular interest, mostly because TGF-β is a major profibrotic cytokine in mesenchymal cells (Massagué 2012). In human skin fibroblasts, TGF-β is an important regulator of collagen homeostasis by stimulating procollagens I and III and reducing MMP-1 transcription. UV irradiation modulates TGF-β pathway at several levels; it decreases type II TGF-β receptor within 4 h in human skin in vivo (Quan et al. 2004), stimulates the intracellular inhibitor of TGF-β signaling Smad-7 (Quan et al. 2005), and reduces levels of connective tissue growth factor (CCN2), an essential mediator of TGF-β effects on collagen synthesis (Duncan et al. 1999; Quan et al. 2002).

Taken together, UV irradiation causes a collagen deficit by shifting homeostasis from production/deposition to degradation (). Because melanin pigments are photoprotective (Kollias et al. 1991), the biological effects of UV irradiation are more pronounced in individuals with light versus dark skin (Fisher et al. 2002; Wang et al. 2008). In lightly pigmented human skin in vivo, transcripts encoding MMP-1, -3, and -9 are induced within 8 h after UV irradiation (Fisher et al. 1996) and enzyme activities are observed 24 h postirradiation in human skin in vivo (Fisher and Voorhees 1998). Similarly, UV irradiation reduces collagen production; type I collagen transcript and protein levels are decreased within 8 h following UV irradiation in human skin in vivo, and remain diminished in the upper dermis 24 h after UV exposure (Fisher et al. 2000). These responses to acute UV irradiation are transient; however, MMP and collagen transcripts normalize to baseline levels by 96 h postirradiation in human skin (Fisher et al. 1996, 2000, 2002).

Accumulation of Fragmented Dermal Collagenous Extracellular Matrix Sustains Collagen Decline in Aged Human Skin

The transiently increased degradation and decreased production of collagen fibrils that occur in response to a single exposure of skin to UV irradiation provides insight into the mechanisms that promote sustained collagen deficit (hypocollagenesis), which characterizes chronologically aged and photoaged human skin. When compared in culture, subject-matched dermal fibroblasts isolated from sun-exposed and -protected human skin produce similar amounts of procollagen I (Varani et al. 2001). This observation suggests that photoaging-induced hypocollagenesis is not related to genetic or intrinsic alterations of dermal cells. Likewise, when compared in culture, isolated fibroblasts harbor modest differences in their metabolism and collagen production (Schneider 1979; Varani et al. 2006), differences that may be specific to the fibroblasts residing in the topmost 10% portion of the dermis (Mine et al. 2008). In any case, the sustained decrease of collagen production in chronologically aged skin measured in vivo appears much more robust than that measured in cultured fibroblasts (Varani et al. 2006). Taken together, these observations support the concept that the dermal microenvironment imposes altered fibroblast functions in chronologically aged and photoaged human skin. Evidence indicates that accumulation of fragmented dermal extracellular matrix is a key factor that mediates many of the characteristic features of aged human skin (see and below).

Accumulation of fragmented collagen in the dermal extracellular matrix leads to sustained reduction of collagen production in chronologically aged and photoaged human skin. (Left panel) In young skin, intact collagen within the dermal extracellular matrix provides attachment sites and mechanical resistance for fibroblasts. Fibroblasts are able to stretch and, under relatively high mechanical tension, show normal collagen homeostasis (collagen production is high, MMP production is low). (Middle panel) On exposure to UV irradiation (photoaging) or oxidative stress (chronological aging), elevated ROS activate signaling cascades that promote reduced collagen synthesis and increased MMP production. Active MMPs cleave the collagenous extracellular matrix, whereas reduced procollagen production limits repair. (Right panel) Accumulation of collagen fragments, which occurs with chronic UV exposure and the passage of time, impairs the mechanical and functional properties of the dermal extracellular matrix. Fibroblasts respond to this degraded dermal extracellular microenvironment by up-regulating MMP expression and down-regulating collagen production, thereby creating a self-sustaining phenotype that promotes skin fragility and age-related diseases.

As detailed earlier, acute UV irradiation induces ROS in human skin. Elevation of ROS levels is also considered a major driving force for natural aging (Harman 1992; Fisher et al. 2009). Transient increase in ROS levels in skin cells, via the mechanisms described above, leads to up-regulation of MMPs, resulting in collagen degradation. Conceivably, because of cross-linking that confers general resistance to further degradation of type I collagen fibril fragments, collagen fibril remnants appear to remain bound within the dermal extracellular matrix. In addition, collagen degradation is accompanied by reduced collagenesis, which likely impedes replacement. Over time, fragmented collagen fibril remnants accumulate and become readily observable in photoaged (Varani et al. 2001, 2002) and aged (Fisher et al. 2002; Varani et al. 2006) human skin dermis.

Histologically, fibroblasts in chronologically aged and photoaged human skin are surrounded by fragmented collagen matrix. These fibroblasts appear to be collapsed, with dramatically reduced cell surface area and fewer contact points with surrounding fragmented collagen fibrils (Varani et al. 2006). Like many other cell types, dermal fibroblasts sense the mechanical load of their environment. Mechanical forces are readily transferred from the extracellular matrix to the cell’s intracellular scaffolding (cytoskeleton), a phenomenon known as mechanotransduction (Chen 2008). The level of mechanical tension within a cell that is bound to its extracellular matrix is determined by at least three factors: (1) the traction forces generated by the cell via its cytoskeleton, (2) the intrinsic resistance of the extracellular matrix, and (3) any externally applied forces to the extracellular matrix transmitted to the cell (Chen 2008). In chronologically aged and photoaged skin, accumulation of fragmented collagen decreases the mechanical resistance of the collagenous extracellular matrix and thereby decreases the mechanical forces within resident fibroblasts. This phenomenon is critical because mechanical forces that control cell shape are intimately coupled, although as yet not fully understood mechanisms, to fibroblast functions. Human dermal fibroblasts respond to low mechanical force by increasing their levels of ROS, reducing collagen production and increasing expression of MMPs (Mauch et al. 1988; Delvoye et al. 1991; Eckes et al. 2006; Fisher et al. 2009). Thus, collagen fragmentation fuels a self-perpetuating cycle of oxidative stress, reduced collagen synthesis, and increased collagen fragmentation in chronologically aged and photoaged human skin ().

The authors acknowledge former and current investigators who have contributed to knowledge of skin aging. Citation of relevant literature could not be comprehensive and apologies are extended to those whose work could not be included in this review. Support was provided by grants AR059678 (L.R.), AG019364 (G.J.F.), and AG031452 (G.J.F.) from the National Institutes of Health.