Topical Platelet Exosomes Reduce Senescence Signaling in Human Skin: An Exploratory Prospective Trial

Supplemental Digital Content is Available in the Text.

Abstract

BACKGROUND

Cellular senescence, an irreversible cell cycle arrest with secretory phenotype, is a hallmark of skin aging. Regenerative exosome-based approaches, such as topical human platelet extract (HPE), are emerging to target age-related skin dysfunction.

OBJECTIVE

To evaluate the cellular and molecular effects of topical HPE for skin rejuvenation after 12 weeks of twice daily use.

METHODS

Skin biopsies were obtained for histological evaluation of senescence markers, p16^INK4a and p21^CIP1/WAF1. Telomere-associated foci, coassociation of telomeres, and DNA damage marker, γH2AX, were assessed. RNA sequencing evaluated senescence associated secretory phenotype (SASP) and extracellular matrix pathways.

RESULTS

p16^INK4a and p21^CIP1/WAF1 staining in senescent skin cells revealed low and high expression subgroups that did not correspond to chronological age. Topical HPE significantly reduced high p16^INK4a cells in the dermis (p = .02). There was also a decrease in telomere damage after topical HPE (p = .03). In patients with high senescent cells at baseline, there was a 40% reduction in proinflammatory SASP. Extracellular matrix remodeling pathways, including collagen and elastic fibers, were up-regulated.

CONCLUSION

Topical HPE, applied on intact skin, reduced senescence signaling and senescence-associated telomere damage after 12 weeks of twice daily use, targeting a path for skin longevity or healthy skin aging.

Skin aging is an inevitable process driven by overlapping intrinsic and extrinsic factors that decrease the skin's structural integrity and physiologic function.^1,2 Skin aging entails dysregulation of skin cells and loss, fragmentation, or fragility of extracellular matrix (ECM) fibers, which are manifested by wrinkling, laxity, and pigmentary abnormalities. Age-related skin changes are the focus of many surgical and nonsurgical treatments aimed at improving overall skin appearance and health.

The expanded hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis.³ Cellular senescence, a defense mechanism to damaging stimuli, has been established as a central pillar of aging, contributing to decline in skin functionality with old age.⁴ Although senescent cells cease to proliferate, they remain metabolically active, secreting factors known as the senescence-associated secretory phenotype (SASP) that influence adjacent, otherwise normal cells to become senescent or dysfunctional.⁵ As such, skin aging is characterized by gradual loss of function and regenerative capacity. Another molecular signature of aging is telomere attrition, or the gradual shortening of the protective telomere caps, which is specifically measured by telomere-associated foci (TAF). Telomere-associated foci are an indicator of genotoxic and oxidative stress that can result in cellular senescence.^6,7

Advancements in the regenerative aesthetics toolkit include exosomes, or extracellular vesicles, that range in size from 40 to 250 nm in diameter with an endosomal origin.⁸ Exosomes can represent a diversity of functions based on their size, content, and cellular origin. Platelets are considered an ideal source for exosome isolation given their propensity for stimulating skin healing.⁹ Human platelet extract (HPE) is a leukocyte-depleted allogeneic exosome product derived from US-sourced, pooled, apheresed platelets with consistent batch purity and potency.⁹ Previously, the author group reported initial findings from this prospective, single-arm, nonrandomized, longitudinal study investigating the safety and effects of topical HPE, the key ingredient containing platelet-derived exosomes, on skin rejuvenation at 6 weeks.^10,11 Phenotypic changes included aspects of facial photodamage such as redness, wrinkles, and melanin production. Herein, the authors evaluate a subset of patients from this study who had skin biopsies after 12 weeks of twice daily topical (plated) Intense, which contains HPE, for in-depth histologic analysis of the cellular senescence profile and its associated secretory phenotype, which collectively affect the aging skin ecosystem.

METHODS

Patient Recruitment

This prospective, single-center, longitudinal study was conducted in accordance with the International Conference on Harmonization, Good Clinical Practice guidelines, Code of Federal Regulations, and the Declaration of Helsinki. Mayo Clinic IRB approved the study (Rochester, MN). All subjects gave informed consent before any study procedures were performed. This study has been discussed with US FDA Center for Biologics Evaluation and Research. Future studies evaluating medical indications and/or skin health parameters will be pursued under FDA Investigational New Drug application. All participants were screened to ensure that they met all inclusion criteria and none of the exclusion criteria before enrolment in the study. Participants recruited met the criteria for age 40 to 85 years, were not pregnant, fully understood the requirements and needs to comply with the study testing, and volunteered willingness to discontinue any other antiaging topical or parenteral treatments for the duration of the study. Participants with mild to moderate global face wrinkles and moderate global fine lines based on modified Griffiths 10-point scale were included. Primary exclusion criteria included participants who received aesthetic or antiaging treatments during the last 6 months, such as dermal fillers, peels, plastic surgery, platelet-rich plasma, or other treatments that could change the skin's surface. In addition, subjects with a dermatologic disease, cutaneous marks on the planned treatment area, skin hyper-reactivity, treatment with prescription-strength vitamin A within the last 3 months, treatment with topical steroids on the treatment area within the past 16 days, and those with a previously observed allergy to colophony, nickel, or food or cosmetic products were excluded. Subjects who met all inclusion criteria and no exclusion criteria were enrolled.

The study subjects followed a standardized twice-daily skin care regimen for the 12-week study evaluation to skin biopsy assessment. A skin punch biopsy was obtained at baseline from the left upper inner arm, and a subsequent skin punch biopsy was obtained at 12 weeks from the right upper inner arm after subjects applied twice daily topical HPE serum. Skin tissue was subject to formalin-fixation with paraffin embedding or flash-frozen for RNA analysis (Table 1).

TABLE 1.

Patient Demographics

Number	Age Mean (SD)	Sex	Fitzpatrick Skin Type
20	54 (11)	Female: 18	I–II: 19
		Male: 2	III–VI: 1

Immunohistochemistry and Telomere-Associated Foci Staining

Immunohistochemical staining of p16^INK4a and p21^CIP1/WAF1 and hematoxylin and eosin was performed on formalin-fixed, paraffin-embedded skin sections of biopsy tissue. Sections were scanned using the Aperio platform, viewed on ImageScope, and quantified using QuPath. Positivity for p16^INK4a and p21^CIP1/WAF1 was reported as percentages of total cells. Subgroups of high versus low proportions of p16^INK4a and p21^CIP1/WAF1+ cells were determined by observing their sample distributions, and thresholds were defined to be 0.26% for p16^INK4a in the epidermis, 1.5% for p16^INK4a in the dermis, and 7% for p21^CIP1/WAF1 in the dermis.

Telomere-associated foci were stained on a randomly selected set of biopsies using an immuno-FISH method, as previously described.⁶ Samples were incubated with rabbit monoclonal anti-γH2AX (1:400, 9,718; Cell Signaling), followed by a goat antirabbit biotinylated secondary antibody (1:200, PK-6101; Vector Labs, Burlingame, CA). After washing, samples were incubated with fluorescein avidin direct conjugate system (1:500, A-2011; Vector Labs), washed, and then cross-linked in 4% paraformaldehyde in phosphate-buffered saline. After washing and dehydration in graded cold ethanol solutions, tissues were air-dried, then denatured in hybridization mix (70% deionized formamide [Sigma], 25 mM MgCl2, 1 M Tris pH 7.2, 5% blocking reagent [Roche] containing 2.5 μg/mL Cy‐3‐labelled telomere‐specific peptide nucleic acid probe [PANAGENE]), and then incubated in a dark, humidified chamber for hybridization. After washing in 70% formamide in 2xSCC followed by 2xSCC and phosphate-buffered saline, tissues were mounted using Prolong Gold Antifade Mount with 4′,6-diamidino-2-phenylindole (a fluorescent stain) (Invitrogen). Sections were imaged using in-depth z-stacking (at least 50 optical slices at ×63 objective). Telomere-associated foci were quantified manually using ImageJ.¹²

RNA-Sequencing and Analysis

Bulk RNA-sequencing data were performed on baseline and treated samples of 8 randomly selected patients. Quality control checks were performed on all the samples using FastQC,¹³ and all samples met quality thresholds. Sequences were preprocessed and aligned to human assembly GRCh38 annotation release 110 using R (version 4.3) packages Rsamtools (version 2.18), GenomicFeatures (version 1.38), GenomicAlignments (version 1.54), and BiocParallel (1.38). Genes with reads of less than 10 transcripts were filtered to improve efficiency and clarity of visualizations.

Next, differential expression analysis was performed using the DESeq2 package in R (version 1.42). After processing, a volcano plot was generated to delineate genes that were up-regulated or down-regulated after topical application, highlighted using a p-value threshold of 0.5 and log2 fold change of 0.6. Next, Gene Set Enrichment Analysis (GSEA)¹⁴ was performed with 1,000 phenotype permutations and no collapse on curated Reactome gene sets (C2). Pertinent pathways were visualized, that is skin-related pathways that were not redundant. Significance was determined by a threshold of false discovery rate q-value <0.25. Heatmaps were generated using the count matrix, and subgroups were analyzed based on proportions of p16^INK4a+ cells observed in immunochemistry, as defined earlier.

Statistics

Twenty participants had 2 samples, 1 collected at baseline, and 1 collected after 12 weeks of topical HPE serum. As such, paired Wilcoxon sign-rank tests were used to compare baseline and treated samples for immunohistochemistry. Similarly, paired Wilcoxon sign-rank tests were used to compare the mean TAF per nuclei in baseline versus treated samples. As this is a pilot study, significance was determined with a threshold of p < .05.

RESULTS

p16^INK4a and p21^CIP1/WAF1 Reduced With Topical Platelet Exosomes in Individuals With High Levels of Baseline Senescence

Immunohistochemical staining of cellular senescence markers p16^INK4a and p21^CIP1/WAF1 was performed to evaluate burdens of senescent cells in skin samples at baseline compared to 12 weeks of topical HPE application. Observing baseline proportions of p16^INK4a and p21^CIP1/WAF1 senescent cells revealed a large range, with 2 subgroups: a subgroup of samples had low proportions of p16^INK4a and p21^CIP1/WAF1+ cells with low variability, and another subgroup of samples had high proportions of p16^INK4a and p21^CIP1/WAF1+ cells with higher variability (Figure 1A–D, See Supplemental Digital Content 1, Figure 1A, B, http://links.lww.com/DSS/B525). Examples of photos from subjects with high proportions of p16^INK4a and p21^CIP1/WAF1 cells, respectively, are shown for clinical correlation (Figure 1A, C). Observed subgrouping is consistent with understanding that individuals with robust immune systems can clear senescent cells and prevent their accumulation, while others fail to clear senescent cells, leading to their accumulation with age. Particularly, a decrease in the proportion of p16^INK4a senescent cells in the dermis was observed after topical HPE (p = .02; Figure 1B). Because of this observation, and the connection between p16^INK4a and chronologic aging in skin,¹⁵ subgroups based on p16^INK4a senescent proportions at baseline were used in further analyses.

Figure 1.

p16 and p21 senescence signaling reduced with topical HPE in subjects with high p16 or p21 at baseline. (A) 52-year-old Fitzpatrick II woman with high proportion of p16+ senescent cells at baseline and post 12-week topical HPE. (B) Percentage of p16+ senescent cells in epidermis and dermis of participants with high proportion of p16+ senescent cells at baseline (0.26% threshold for epidermis and 1.5% for dermis). (C) 67-year-old Fitzpatrick II woman with high proportion of p21+ senescent cells at baseline and post 12-week topical HPE. (D) Percentage of p21+ senescent cells in dermis of participants with high proportion of p21+ senescent cells at baseline (7% threshold). HPE, human platelet extract.

Telomere Damage Reduced With Topical Platelet Exosomes

To evaluate another hallmark of aging, telomere attrition was evaluated by immuno-FISH of was displayed (Figure 2A). Specifically, TAF are imaged microscopically and were determined as overlap between a stain for telomeres and γH2AX, a DNA damage associated marker (Figure 2B). Overall, there was a decrease in the number of TAF in each cell nucleus after topical HPE (p = .03; Figure 2C). The decrease in TAF in each nucleus could be observed across the distribution of number of TAF per nucleus (See Supplemental Digital Content 1, Figure 2C, http://links.lww.com/DSS/B525).

Figure 2.

Telomere damage reduced with topical HPE. (A) 41-year-old Fitzpatrick II woman with high TAF at baseline and post 12-week topical HPE. (B) Example of TAF imaged in the epidermis with a TAF in a nucleus highlighted by the white triangle. (C) Average TAF per nuclei in sample of baseline versus post-topical HPE. (D) ECM gene expression in patients with high proportion of p16 senescent cells, baseline versus topical HPE. ECM, extracellular matrix; HPE, human platelet extract; TAF, telomere-associated foci.

Extracellular Matrix Synthesis and Keratinization Pathways Increased, While Inflammatory Pathways Were Modulated With Topical Platelet Exosomes

Bulk RNA-sequencing was performed on a randomly selected sample of subjects to broadly evaluate the effects of the 12-week topical HPE. First, differential expression analysis was performed to identify genes significantly up-regulated or down-regulated after topical HPE (See Supplemental Digital Content 1, Figure 1A, http://links.lww.com/DSS/B525). Some of the significantly up-regulated genes included GPRC5A, related to epithelial cell differentiation, EGFL6, potentially related to hair follicle morphogenesis and matrix assembly, and KRT33A, related to keratinization.

Further, GSEA was performed to identify pathways enriched or down-regulated by the 12-week topical HPE (see Supplemental Digital Content 1, Figure 1B, http://links.lww.com/DSS/B525). Many significantly up-regulated pathways were involved in keratinization and ECM remodeling, including collagen, proteoglycans, and elastic fibers. Some additional up-regulated pathways were related to platelet function, as expected from the source of topical HPE product.

Due to cellular aging changes observed in p16^INK4a and p21^CIP1/WAF1 in patients with high proportions of p16^INK4a and p21^CIP1/WAF1 senescent cells at baseline, proinflammatory senescence-associated secretory phenotype (SASP) factors were evaluated. Decreases in expression of some proinflammatory SASP factors were observed in patients with high proportions of p16^INK4a senescent cells at baseline (see Supplemental Digital Content 1, Figure 1C, http://links.lww.com/DSS/B525). Reductions in these proinflammatory cytokines suggest that topical HPE not only affected senescent cells but also their proinflammatory effects.

To further investigate changes in ECM synthesis, a collection of collagen and elastic fiber–related genes were examined (see Supplemental Digital Content 1, Figure 1D, http://links.lww.com/DSS/B525). In the subgroup of subjects with high proportions of baseline p16^INK4A senescence, widespread up-regulation of collagen and elastin-related genes were observed. A trend toward up-regulation of collagen and elastin-related genes were also observed in the subgroup of subjects with low proportions of baseline p16^INK4A (see Supplemental Digital Content 1, Figure 2D, http://links.lww.com/DSS/B525).

Discussion

Skin aging, associated with a time-dependent functional and structural decline, has piqued the quest to slow or reverse biological aging in aesthetics.^4,16 Akin to organismal whole-body aging, the skin is subject to gradual loss of function and regenerative capacity with age.¹⁷ In this process, cellular senescence directly plays a damaging role by impairing tissue regeneration, causing inflammation and fibrosis.¹⁸ Indeed, the rise of regenerative interventions, such as topical platelet extracellular vesicles or exosomes, provide a new approach to target root-cause reparative strategies at the cellular and molecular levels.¹⁹

Results of this study highlight the substantial heterogeneity in the senescent cell burden in human skin samples at baseline, which influenced subsequent responses to topical platelet exosomes. The distinct subgroups within the authors' patient population characterized by varying initial proportions of baseline senescent cell burden suggest differential abilities to clear these cells, possibly linked to variations in immune function. Notably, individuals with higher baseline senescent burden exhibited greater responses to the treatment, including a significant reduction in p16 senescent cells after topical HPE. A possible explanation is that platelet exosomes can polarize toward a regenerative immune response,²⁰ increasing their ability to clear senescent cells. These findings underscore the potential role of intervening in cases of premature aging. Indeed, baseline senescent cell profiles may serve as predictive biomarkers to assess prolongevity outcomes in future interventions aimed at mitigating cellular senescence in skin aging.

Furthermore, the authors' investigation into telomere damage using TAF provided insights into the effects of topical HPE on this molecular feature of aging, as telomeres traditionally protect chromosomal ends. At baseline, the authors observed considerable variability in TAF levels among subjects, indicative of diverse degrees of telomere damage.²¹ Indeed, targeting cellular senescence and aberrant senescent signaling can mitigate this telomere attrition.^22,23 After topical HPE, a significant reduction in TAF per nucleus was evident (p = .03). These findings suggest that topical platelet exosomes may have a protective effect on telomeres, potentially slowing cellular aging, or promoting clearance of senescent cells induced by telomere damage. Study limitations include the lack of placebo-control group, limited skin of color subjects for generalizing study findings, and small sample size.

Moreover, RNA-sequencing analysis provided comprehensive insights into the molecular effects of the 12-week topical HPE serum on skin aging markers and associated pathways. Sequencing results highlighted enrichment in pathways related to keratinization, ECM remodeling, and platelet function, consistent with exosome source. Importantly, in participants with high baseline proportions of senescent cells, a notable decrease in SASP factors was observed after topical HPE, indicating a reduction in the proinflammatory milieu associated with senescent cells. Furthermore, enhanced expression of collagen and elastic fiber–related genes in participants with high baseline senescent levels suggests a targeted effect of the topical HPE on ECM synthesis. These findings collectively underscore the multifaceted impact of topical HPE on molecular pathways implicated in skin aging, emphasizing its potential to mitigate age-associated changes at the cellular level. Further research is warranted to validate these observations and elucidate their clinical implications for long-term skin rejuvenation therapies.

Conclusions

Skin aging represents a complex interplay of biological processes marked by functional and structural decline over time. This study evaluated the role of topical HPE to assess the role of exosomes in mitigating senescence-associated changes and telomere damage. These insights into emerging regenerative strategies pave the way for future research into personalized antiaging therapies targeting cellular senescence and the biological clock.