Characterization of Skin Aging–Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin

Abstract

Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin

“…In relation, Waldera‐Lupa et al . recently reported that intrinsically aged fibroblasts exhibited more frequently nuclear foci positive for two common features of DNA damage (p53‐binding protein 1 and promyelocytic leukaemia protein).…”

“…Of interest, the recently described age‐related changes in the secretome and cytokine release profile of fibroblasts from young and old donors indicated that fibroblasts isolated from intrinsically aged skin exhibited some, but not all, molecular hallmarks of replicative senescence. Indeed, they secreted a specific unique pattern of protein, distinct from the classical SASP, with evidence of 27 proteins involved in the biological processes of ‘metabolism’ and ‘adherent junction interactions’.…”

“…This is evidenced in the Table S1, where the main hallmarks of fibroblast ageing are summarized. Recently, some differences between intrinsically aged and replicatively aged human dermal fibroblasts have been reported in two articles . A recent literature overview by Tigges et al .…”

Sirtuins and Accelerated Aging in Scleroderma

“…Aging and fibrosis share molecular mechanisms in multiple organs, including the lung [7, 8], heart [9, 10], vascular wall [11], kidney [12], liver [13, 14], and skin [15, 16]. Accumulating literature indicates that SIRTs regulate pathways and molecules involved not only in aging, but also in tissue and organ fibrosis (Table 1).…”

The Senescence-Associated Secretory Phenotype: Critical Effector in Skin Cancer and Aging

“…Thus, the increased numbers of senescent cells and the resulting SASP in human tissues with aging have long provided a potential mechanistic link for this connection, as the SASP can lead to increased cancerous proliferation both in vitro and in vivo (Krtolica et al, 2001). Indeed, intrinsically aged, yet nonsenescent, human skin fibroblasts were recently shown to express numerous SASP genes, including IL1β, IL6, MMP1, MMP3, MMP10, SERPINB2 , CXCL10 , AREG , fibroblast growth factor-2, tumor necrosis factor-α, and vascular endothelial growth factor (Waldera Lupa et al, 2015). This underlying inflammatory microenvironment in aging and other settings of DNA damage may ultimately promote carcinogenesis in the skin (Figure 2).…”