ceAF supports cell growth and promotes wound healing in vitro
Given that ceAF is a source of embryonic stem cells, we believed that ceAF comprised biologically balanced secretory growth factors, cytokines and other ligands collectively dubbed proliferation-stimulants and, as a prelude to exploring its therapeutic potential, tested its ability to support the growth of cultured HeLa cells using FBS as a control. Starting from ~20% confluence, HeLa cells were cultured in DMEM supplemented with increments of ceAF (0-20%, v/v) or 10% FBS for 48 hours; 5% ceAF optimally, and nearly as potently as 10% FBS, supported cell growth that roughly underwent 2 doubling times (Figure 1A/1B). Given that cultured HeLa cells exhibited elevated p16 expression in higher doses of ceAF (20% in particular; Figure 1B bottom panel), we proposed that the lower efficacies of higher ceAF doses, as compared to 5% ceAF, for supporting cell growth (Figure 1B upper panel) were attributable to an anti-proliferative activity of p16. This suggests a manifestation of attenuating activities with higher doses of ceAF and emphasizes an importance of balanced (or optimal) dose of ceAF in its application on wound healing.
Prompted by the above, we established an in vitro wound healing model utilizing HaCaT cells, representing an epidermis origin (12); given fastidious nature of ceAF, we employed 5% and 10% doses in our later efforts on the presumption that one or a dose somewhere in between works optimally. Scratches were made on near-confluent monolayers and cells treated with ceAF. Agreeing with cell proliferation assays, 5% ceAF healed the scratches better than 10% ceAF within 24 hours; ectopically expressing p16 hindered the healing in vitro (Figure 1C/1D; also see below).
Encouraged by the above in vitro results, we examined the healing capacities of ceAF on surgically-made cutaneous wounds in a murine model. Mice treated with 5% ceAF healed most expeditiously, manifesting virtually scar-less skin in 10 days (Figure 2A/2B). Dermal application of naturally produced/topically administered exogenous p16 has been a practice in wound healing research, given that this small-size protein can be up-taken by G-protein coupled receptors (13, 14). In our hands, p16 when co-applied within a dosage employed by other practices significantly attenuated the wound healing process, which served an offset for facilitated wound healing brought about by ceAF particularly at a 5% dose (Figure 2A/2B). This is consistent with results of the in vitro model (Figure 1C/1D).
p16 impedes wound healing in vitro and in vivo
Under stresses such as those that induce senescence, p16 expression is up-regulated; p16 was reported to be able to drive senescence-associated secretory phenotypes (SASP) that provoke chemokine/cytokine pathways in the inflammatory phase of wound healing preluding the proliferative phase. SASP factors exert functions transiently, but whether this “transient senescence” is p16-dependent remains unresolved. To examine whether p16 expression in conjunction with ceAF facilitates wound healing, we ectopically expressed Flag-tagged p16 in HaCaT cells supplemented with either 5% or 10% ceAF; p16 unexpectedly and markedly retarded the closure of scratched areas (Figure 1C/1D) and cellular proliferation (see below). We had serially titrated down p16-expressing plasmid in transfection assays, and were unable to identify a dose in which ectopic p16 expression would additively or synergistically facilitate wound healing by ceAF (data not shown). In the animal models, similar patterns were observed whereby wound healing was attenuated when p16 was co-daubed with ceAF (Figure 2A/2B). Taken together, these results suggest that, at least in our assay systems, the signalling involving SASP factors and transient senescence might not be strictly dependent on p16. Other pathway(s) such as, notably, a p21-mediated pathway may compensate for and/or play redundant roles in this program (see below).
ceAF facilitates wound healing in vivo and manifests increased collagen density and thickened epidermis on healed wounds
On day 10, we observed prominent contraction on the ceAF-daubed wounds particularly with a 5% dosage, and significant re-epithelialization (Figure 2A) [significance of 5% ceAF as compared to other groups summarized in Table S1(A)]. However, mice treated with ceAF+p16 exhibited wounds with wide epithelial and dermal edges with the gap comprising necrotic fibrinoid, cell debris, intense inflammation and angiogenesis plus reduced granulation tissues and significant lower deposition of collagen as judged by the trichrome staining (Figure 2B), suggesting a wound healing that was hampered by p16. Furthermore, histological (H&E) staining showed that, in the 5% ceAF group, wound sections showed a neoformative epidermal layer that was remarkably thicker (Figure 2C), indicating dense epidermal ridges, sebaceous glands, cellular layers and more formation of primitive hair follicle structures. On the contrary, the ceAF+p16 group wounds exhibited much lower migration of endothelial cells and fibroblasts as well as significantly reduced epidermal thickness of the skin with impaired granulation tissues (Figure 2C), again indicating a wound healing hampered by p16.
ceAF facilitates an S to G2/M transition and cell migration that is offset by p16
To test whether the healing process facilitated by ceAF and attenuated by p16 expression or administration (Figures 1/2) were related to cell cycle progression, we cultured HaCaT cells with ceAF (5% or 10%) with or without ectopic p16 expression and subjected cells to FACS analyses. Cells in the 5% ceAF group exhibited highest percentile of G2/M cells, but p16 expression on top of that retarded quite some cells in the S-phase that had obvious difficulty to enter the G2 phase (Figure 3A), suggesting that the wound-healing-promoting (by ceAF) and the wound-healing-attenuating (by p16 expression/administration) phenotypes (Figures 1/2) are at least in part due to their functions exerted on cell cycle progression.
Cell migration also plays important roles in wound healing (15-17) ; we performed trans-well migration assays using HaCaT cells. The 5% ceAF-treated group exhibited a high number of trans-well-passed cells; likewise, and consistent with the cell cycle results (Figure 3A), expressing p16 on top of ceAF significantly attenuated trans-well passaging (Figure 3B).
ceAF triggers transient senescence
Senescence is generally recognized to be a chronic mechanism associated with, e.g., age-related pathologies; but tissue damages such as skin wound can induce, albeit transiently, senescence dubbed transient senescence . This might be an evolutionarily acquired mechanism; immune surveillance later clears the transiently senescent cells, which manifest certain levels of Senescence Activated β-galactosidase (SA-β-gal) activity that can be detected histochemically, scoring both numbers of positive cells and intensities. Thus, SA-β-gal is a good biomarker for senescent cells in both cell culture and in vivo.
We examined SA-β-gal expression patterns in HaCaT cells incubated in control vs. ceAF-supplemented media with or without a p16 ectopic expression (Figure 4A). The number of SA-β-gal+ cells was highest in the 5% ceAF group (Figure 4B; upper panel), but more intense SA-β-gal+ signals were observed in cells ectopically expressing p16 (Figure 4B; bottom panel); both criteria were deemed statistically significant. Given that 5% ceAF exerted optimal functions on wound healing and expediting cell division (Figures 1-3), we propose that a proper (homeostatic) SA-β-gal expression level indicates a transient senescence that facilitates wound healing, while a p16-intensified SA-β-gal activity may represent senescence at a more permanent scale that is above a threshold and, based on our earlier data, hampers wound healing and blocks cell cycle.
Expression profiling of factors involved in cutaneous wound healing
Inflammation, proliferation and remodelling are three sequential yet overlapped phases of wound healing; upon wounding and thereafter, diverse chemokines, cytokines and growth factors are locally enriched and distantly attracted at wound sites, a signalling cascade triggered by SASP factors transiently released by transient senescent cells. They in turn sequentially or concomitantly orchestrate a healing program. For instance, involving diverse kinds of cells (e.g., epidermal and endothelial cells, fibroblasts, keratinocytes, neutrophils etc) and acting paracrinally, these factors comprise a host of chemokines that recruit pro-inflammatory cells including macrophages, which in turn release (additional) cytokines/growth factors to facilitate wound healing (18, 19).
We employed wounded/repaired skin tissues from animals (in vivo samples) and HaCaT cells (in vitro samples) treated under specified conditions and built an expression profile of cellular factors as afore-emphasized to be involved in tissue repair. In vivo samples (collected at day 10) were used to examine the expression pattern of genes encoding proteins that dictate the integrity of restored skin boundary, i.e., degree of remodelling. As seen (Figure 4C, quantification; Figure 4D, Heatmap analyses), mRNA expression levels of three tested genes, i.e., that of keratin, keratin- 10 and endothelin, were significantly up-regulated in the 5% ceAF group (to a lesser degree in the 10% ceAF group); p16 played an offset role (the control group here was the wound daubed with PBS/40% glycerol). In conjunction with the data shown below, we propose that ceAF plays multi-complex roles to facilitate wound healing.
We also established an expression profile of 12 of ~25 SASP factors reported (20, 21) using HaCaT cells incubated with FBS vs. ceAF, with or without ectopic p16 expression. Of the tested SASP factors, the expression of all, save p16, were up-regulated significantly in cells incubated in 5% ceAF and to a lesser degree 10% ceAF (Figure 4C, quantifications normalized against a house-keeping gene; Figure 4D, Heatmap analyses); matter-of-factly, p16 played offset roles on ceAF-enhanced expression of tested genes, and the ostensible variations of p16 expression per se (Figure 4C; last panel) were most-likely due to the presence/absence of an ectopic p16 gene.
Physiological interpretation of a ceAF-mediated expression profile
Structural proteins: For normal skin physiology, the epithelial keratinization is of fundamental importance for counteracting mechanical stress and protecting against pathogenic invasion, and endothelin was suggested to be involved in anabolism of collagen and related proteins (22, 23). In addition, endothelin is a mediator for morphogenesis in other systems including bone regeneration and skeleton formation (24). Thus, elevated expression levels of keratin, keratin-10 and endothelin in the cutaneous wound healing model reflect a more completed remodelling phase.
Interleukins, chemokines and growth factors: Interleukins serve key modulators for inflammatory responses including that in skin repair, and is involved in activation, differentiation and proliferation of endothelial cells, fibroblasts, keratinocytes and leukocytes(25). For instance, IL-6 is released as an early response to tissue injury to induce a signalling of pro-inflammatory cytokines from the resident macrophages and stromal cells (26). In addition, IL-1A is an autocrine regulator for basal keratinocytes proliferation inside the bulge region of hair follicles and an integral agent of the epidermal stem cell population (27); chemokines CCL5/CCL2 can function as attractants for local and distant macrophages to be enriched at wound sites. Furthermore, growth factors such as TGF-b, VEGF, PDGFA/B can regulate direct (cell-cell) and indirect (paracrine) linkage between different cell types and cellular basement and, more relevant to this work, are involved in restoring skin boundary integrity. Therefore, these factors conceivably function at different healing stages covering the inflammatory and proliferative phases and possibly even the remodelling phase. Emphasizing important roles on wound healing, the expression of these factors is quite prominently up-regulated in ceAF-treated cells and offset by p16 (Figure 4C-D).
PAI-1 and vimentin: Plasminogen activator inhibitor type 1 (PAI-1) is expressed on the surface of keratinocytes, its activation enhances cellular proliferation (18). PAI-1 limits plasmin generation to sustain cell migration and proliferation, a key indicator for re-epithelialization of keratinocytes (28). The intermediate filament protein vimentin plays significant role for the “epithelial to mesenchymal transition (EMT)”, and studies reveal that vimentin participates in a number of cellular processes including migration and invasion of cells, cell adhesion, cytoskeletal rearrangements, signalling, plasticity and regulation of cell morphology (29, 30). These proteins in principle are largely involved in the remodelling phase of a wound healing program.
Cell cycle/senescence regulators: p16 and p21 are well known proteins in regulating cell cycle and senescence (31, 32); as far as our assay systems are concerned, p16 is, as opposed to a recognition supported by certain experimentation, not playing a role in establishing the transient senescence that readies wounds to undergo healing. From all criteria, p16 is actually an offsetting factor for therapeutic values of ceAF. Our data (Figure 4C; second last panel) support a notion that p21 might as well play compensatory role(s) by itself or in conjunction with other factor(s), e.g., a non-redundant function afforded by the third component of the INK4-ARF locus, INK4B.
Further Discussion and Perspectives
Intrinsic to and secreted by developing chick embryos 6-8 days post fertilization, ceAF exhibits powerful wound healing capacities apparent in mammalian cutaneous wound healing models, in which ceAF with no preservatives/fortification displays potent efficacy both in vitro and in vivo. Naturally, ceAF provides nourishment and protection against environmental stresses including pathogens, and is thought to possess diverse signalling molecules at biologically homeostatic levels to support embryogenesis. This view is especially important given a manifestation of the attenuating activities at high doses (Figure 1B). Of note, given that the control for establishing the expression profiling with culture cells (Figure 4C/D) was FBS, ceAF must contain components above and beyond ordinary growth factors and/or other molecules that solely support cell growth and migration as revealed in Figure 1A/B and Figure 3B. Recently, occurrence of senescence and roles of SASP factors during embryogenesis (33) and in limiting fibrosis upon tissue injury (34, 35) were reported. Thus, scar-less tissue repair might take place in embryos, supporting our postulation that ceAF comprises a set of evolutionarily conserved, at least among vertebrates, factors involved in wound healing.
Conceivably, at least in our assay systems and as in many signalling cascades, signals from ceAF, however chick-embryo-derived, obviously can be received and amplified by cells in the wound/surrounding areas of a murine model to facilitate the wound healing. From a biochemical, mechanistic and therapeutic perspective, chromatographic isolation of active fractions as well as reconstitution to formulate an efficient cocktail starting from ceAF and, probably much more importantly, efforts to rid of attenuating activities (Figure 1B) also by biochemical isolation, are a daunting challenge yet obviously a worthy goal.