Senescent dermal fibroblasts accumulate and secrete chemically reactive lipids that are components of the senescence-associated secretory phenotype (SASP). These lipids, including 4-hydroxynonenal (HNE) and reactive oxidized phospholipids (OxPL), covalently bind to and modify proteins via Schiff base formation or Michael adduction. Our study examined lipid-induced collagen modifications and their impact on skin cells to evaluate the long-term consequences of senescent cells on the tissue microenvironment. Using mass spectrometry and biochemical analyses, we identified both high and low molecular-weight modifications to collagen types I, II and IV. Collagen modified by HNE reduced fibroblast proliferation and induced stress responses. In contrast, collagen modified by OxPL provoked inflammatory signaling. Both types of modifications influenced matrix remodeling by increasing proteinase expression while reducing collagen expression. Modified collagen also elevated levels of intracellular reactive oxygen species and lipid peroxidation. Macrophages cultured on modified collagen displayed altered cytokine profiles and Toll-like receptor signaling impairment, that depended on the specific type of lipid modification. Similarly, keratinocytes exposed to modified basal lamina collagen IV showed transient stress responses, increased cytokine expression, and reduced matrix metalloproteinase expression. Furthermore, lipid-modified collagen incorporated into organotypic skin equivalents disturbed keratinocyte differentiation and elevated markers of cellular senescence. These skin models also showed reduced epidermal thickness with HNE-modified collagen and parakeratosis on OxPL-modified matrices. In conclusion, the findings suggest that SASP lipids secreted by senescent fibroblasts alter collagen structure and the fate of residing cells. The responses are likely caused by cell-associated oxidation events upon interaction of cells with a lipid-modified matrix and can be inhibited by antioxidants in macrophages. Given collagen's long half-life in tissues, these modifications may represent a persistent mechanism by which senescent cells affect the tissue microenvironment beyond the lifespan of soluble SASP factors - thereby sustaining an aged phenotype over extended periods.

APOSECTM, a complex mixture of secreted proteins, lipids, and exosomes from stressed peripheral blood monocytes, is currently in clinical trials for the treatment of chronic wounds. When applied to open wounds, 1 mL reconstituted APOSECTM lyophilisate is syringe-mixed with 3 g sterile hydrogel prior to administration. This study investigates the pharmaceutical performance of this novel administration system. A gel formulation (APOgel) was developed for terminal sterilisation in pre-filled syringes with post-sterilisation viscosity (∼325-350  Pa·s at 1  s-1) comparable to a commercial benchmark gel. Syringe mixing of APOgel with a liquid APOSECTM surrogate (3:1) reduced viscosity by ∼ 67% but was highly reproducible across different operators (CV < 6%). Administration of three sequential dose units of the mixture from the syringe revealed an ∼ 20% higher content of active ingredients in the first and final dispensed compared to the middle unit, indicating non-uniform mixing in the closed syringe system. In vitro release studies over 72  h showed a 32% and 48% higher release of a small molecule marker and total proteins from the sterile APOgel compared to the benchmark, while total release at 72 h was 100% for small molecules and 25% for total proteins from the APOgel system. Efficacy studies in a murine wound healing model showed no significant difference between APOgel and the benchmark. Overall, APOgel shows potential as a hydrogel platform for topical administration but its mixing system, release kinetics, and test methods could benefit from further optimisation before broader use.

Epidermal keratinocytes (KCs) undergo a tightly orchestrated differentiation programme that culminates in the formation of a protective barrier between the body and the environment. Although this process has been studied extensively, several regulators that coordinate KC survival, stratification, and terminal differentiation remain incompletely defined. To identify mechanisms governing epidermal homeostasis, we compared transcriptomic profiles of undifferentiated KCs, KCs differentiated in monolayer culture, and fully stratified KCs in three-dimensional organotypic skin equivalents. Both differentiation models showed downregulation of cell-cycle-associated transcripts and induction of KC differentiation genes, whereas genes involved in extracellular-matrix organisation were preferentially induced in skin equivalents. Transcription-factor activity inference based on target gene expression identified the transcriptional repressor and ubiquitin/SUMO E3 ligase TRIM28 as a candidate regulator of epidermal homeostasis. Single-cell RNA sequencing and immunostaining of human epidermis revealed strong TRIM28 expression throughout the living epidermal layers, with marked loss in the outermost terminally differentiated compartment. Functional depletion of TRIM28 in primary human KCs impaired epidermal development, increased apoptosis, and produced markedly thinner organotypic epidermis despite preserved stratification. TRIM28-deficient skin equivalents showed limited transcriptional reprogramming associated with reduced p53 ubiquitination, p53 accumulation, and apoptosis of basal KCs. Collectively, these findings identify TRIM28 as an important candidate regulator of epidermal homeostasis and suggest that TRIM28 supports epidermal expansion, at least in part, by contributing to p53 turnover.

The human thymus plays a key role in the development of the adaptive immune system. Its development and pathologic aberrations with missing involution occupy the scientific world for years. Here, we present a comprehensive single-cell RNA sequencing (scRNA-seq) analysis of 453,727 cells across 53 datasets derived from healthy prenatal, pediatric, and adult thymic tissues, as well as six pathological conditions, including thymic hyperplasia and thymic epithelial tumors (types A, AB, B, C, and micronodular thymoma). We created a high-resolution cellular atlas revealing disease-specific cellular populations and transcriptional programs, particularly within fibroblast subsets and thymic epithelial cells. Comparative analysis uncovers distinct intercellular communication patterns and identifies transcriptional alterations associated with thymic pathology. Integration with published bulk RNA-seq datasets supports the robustness and translational relevance of our findings. This study provides a foundational resource for understanding the cellular and molecular landscape of thymic development and disease, offering avenues for diagnostic and therapeutic innovations.

Changes in brain iron levels are a consistent feature of multiple sclerosis (MS) over its disease course. They encompass iron loss in oligodendrocytes in myelinated brain regions and iron accumulation in myeloid cells at so-called paramagnetic rims of chronic active lesions. Here, we explore the mechanisms behind this overall shift of iron from oligodendrocytes (OLs) to myeloid cells (MCs) and the loss of total brain-iron in MS. We investigated the expression of various iron importers and exporters, applying immunohistochemistry to a sample of control and MS autopsy cases. Additionally, we studied the transcriptional response of iron-related genes in primary rodent OL progenitor cells (OPCs) and microglia (MG) to various combinations of known MS-relevant pro-inflammatory stimuli together with iron loading. Histologically, we identified a correlation of OL-iron accumulation and the expression of the ferritin receptor TIM1 in myelinated white matter and observed an increase in the expression of iron-related proteins in myeloid cells at the lesion rims of MS plaques. qPCR revealed a marked increase of the heme scavenging and degradation machinery of MG under IFN-γ exposure, while OPCs changed to a more iron-inert phenotype with apparent decreased iron handling capabilities under MS-like inflammatory stimulation. Collectively, our data suggest that OL iron loss in MS is mainly due to a decrease in ferritin iron import. Iron accumulation in MCs at rims of chronic active lesions is in part driven by up-regulation of heme import and metabolism, while these cells also actively export ferritin.

Recent literature gives different results on morbidity and mortality after COVID-19 hospitalization as compared to Influenza. In this registry-based study in Austria, we compared the short- and long-term outcomes after COVID-19 or Influenza hospitalization and associations with their baseline medication load. Data were provided on children and adolescents hospitalized with COVID-19 (sample size: 1061) in the years 2020 and 2021 or with Influenza in 2016-2021 (sample size: 2781) as well as on matched controls from the Austrian population (10,626 controls for COVID-19 and 27,634 for Influenza). The median follow-up time was 430 days in the COVID-19 and 1221 days in the Influenza group. Hospitalized children were more likely to have a larger disease burden as compared to the general population. Influenza patients were observed to be generally younger with a larger percentage of polypharmacy than those with COVID-19. No significant difference in the time to hospital discharge was found between Influenza and COVID-19 patients (HR 1.22 [95% CI 0.97-1.55], p = 0.093). The risk for readmission was significantly higher for Influenza (HR 1.23 [95% CI 1.03-1.47], p = 0.021). In-hospital mortality (COVID-19: 0.94%; Influenza: 0.22%) and 1-year mortality (COVID-19: 1.13%; Influenza: 0.31%) were observed to be higher in COVID-19 patients but severe events were generally rare. The findings suggest that COVID-19 should not generally be considered a milder disease than Influenza.

Background/Objectives: Soluble ST2 (sST2) has gained recognition as a clinically relevant biomarker across a spectrum of inflammatory, cardiovascular, and respiratory conditions. However, the lack of assay standardization raises concerns about result comparability across platforms and studies. Methods: This study systematically evaluated serum sST2 concentrations measured with two ELISA systems-DuoSet and Quantikine-produced by the same manufacturer (R&D Systems, Minneapolis, MN, USA). Results: Using archived serum samples from healthy volunteers and marathon runners, we identified marked discrepancies: serum sST2 concentrations using the DuoSet recombinant standard were on average 4.3-fold higher than those using Quantikine (median 308.3 [106.6-608.6] vs. 71.5 [41.8-115.6] ng/mL). On the pre-coated Quantikine plate, using the DuoSet recombinant standard increased calculated concentrations 4.3-fold compared with the native Quantikine standard (median 308.3 [106.6-608.6] vs. 71.5 [41.8-115.6] ng/mL). On the manually coated DuoSet plate, the DuoSet standard yielded higher medians than the Quantikine standard (8.0 [5.6-11.3] vs. 5.0 [3.7-7.4] ng/mL). Furthermore, between-lot variability within the same ELISA platform resulted in concentration shifts from 0.09 [0.07-0.10] ng/mL (2016) to 1.17 [0.81-3.23] ng/mL (2023) using the same sample. Previously published studies also exhibited wide inter-study variability among healthy cohorts. Conclusions: These findings emphasize that current ELISA systems for sST2 are not standardized and that cross-study comparisons should be interpreted with caution. Until universal standardization is implemented, sST2 should primarily be used for within-study comparisons. This variability may limit the reliability of longitudinal sST2 assessment even in clinical settings.

Platelet-rich fibrin (PRF) and Enamel Matrix Derivatives (EMD) can support the local regenerative events in periodontal defects. There is reason to suggest that PRF and EMD exert part of their activity by targeting the blood-derived cells accumulating in the early wound healing blastema. However, the impact of PRF and EMD on blood cell response remains to be discovered. To this aim, we have exposed human peripheral blood mononucleated cells (PBMCs) to PRF lysates prepared by a swing-out rotor and EMD, followed by bulk RNA sequencing. A total of 111 and 8 genes are up- and down-regulated by PRF under the premise of an at least log2 two-fold change and a minus log10 significance level of two, respectively. Representative is a characteristic IFN response indicated by various human leukocyte antigens (HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DRA, HLA-DRB1, HLA-DRB5), gamma Fc receptors (FCGR1A, FCGR1B, FCGR3B), chemokines (CXCL9-11), and calprotectin (S100A8/9 and S100A12), complement (C1QA/B, C2) and interferon-induced guanylate-binding proteins (GBP1, GBP5). With EMD, 67 and 29 genes are up- and down-regulated, respectively. Characteristic of the upregulated genes are tensins (TNS1 and TNS3). Among the genes downregulated by EMD were epsilon Fc receptors (FCER1A; FCER2), Fc receptor-like proteins (FCRL1, FCRL3) and CX3CR1. Genes commonly upregulated by PRF and EMD were most noticeably NXPH4 and MN1, as well as FN1, MMP14, MERTK, and AXL. Our findings suggest that PRF provokes an inflammatory response, while EMD dampens IgE signaling in peripheral mononucleated blood cells.

Merkel cell carcinomas (MCCs) are rare, highly aggressive skin cancers with poor outcome due to early lymphatic tumor spread and frequent recurrences. MCCs mostly occur in the head and neck and are mainly caused by an infection with the Merkel cell polyomavirus (MCPyV). Increasing evidence suggests that Piwil-2 and small non-coding PIWI-interacting RNAs (piRNAs) play an important role in solid malignancies and we thought that this might also be the case for MCCs. Therefore, Piwil-2 expression was first evaluated in 27 MCC specimens and correlated with oncological outcome. We found an association with high Piwil-2 expression and advanced tumor stage, MCPyV positivity and poor outcome. Next, we utilized siRNAs for Piwil-2 knock-down in MCC cells. Downregulation of Piwil-2 caused a significant change of 202 different piRNAs and 419 proteins. Interestingly, proteins related to viral driven MCC pathways (TRRAP, BRD8, PRIM2, ORC4) were significantly downregulated. Moreover, there was a moderate cell cycle arrest of cells in the G0/G1-phase, as well as a significant upregulation of SOX-2, a key regulator of Merkel cells. Altogether, Piwil-2 poses a poor prognosticator in MCCs, which is linked to MCC oncogenesis and SOX-2. Further research is needed to better understand underlying mechanisms and to prove their clinical relevance.

Burn injuries often lead to severe complications, including acute respiratory distress syndrome (ARDS), driven in part by systemic inflammation and glycocalyx disruption. In this study, we analyzed the sera of 28 patients after burn trauma and utilized single-cell RNA sequencing (scRNA-seq) along with microarray transcriptomic analysis to decipher the impact of burn injury on glycocalyx derangement. We observed the significant upregulation of immune cell-derived degrading enzymes, particularly matrix metalloproteinase-8 (MMP8), which correlated with increased immune cell infiltration and glycocalyx derangement. Serum analyses of burn patients revealed significantly elevated levels of shed glycocalyx components and MMP8, both correlating with the presence of inhalation injury. Consequently, the treatment of human in vitro lung tissue models with MMP8 induced significant glycocalyx shedding in alveolar epithelial cells. Together, based on these findings, we propose that MMP8 plays a previously unrecognized role in glycocalyx disruption and subsequent lung injury post-burn, which implies that inhibiting MMP8 may represent a promising therapeutic strategy for alleviating lung injury after burn trauma.

Marathon running exerts physical stress and may lead to transient immune dysregulation, increasing susceptibility to airway inflammation and exercise-induced bronchoconstriction (EIB). This study investigated systemic levels of antimicrobial peptides in athletes and their association with EIB. Serum concentrations of angiogenin, human beta-defensin 2 (hBD-2), major basic protein (MBP), S100A8, and S100A8/A9 were measured in 34 marathoners and 36 half-marathoners at baseline, immediately after a race, and seven days postrace using enzyme-linked immunosorbent assays and compared with 30 sedentary controls. Lung function was assessed by spirometry to identify bronchoconstriction. Levels of hBD-2 and S100A8/A9 were significantly elevated postrace in runners compared to baseline and controls, returning to baseline during recovery. During recovery, S100A8 levels remained slightly elevated in marathoners with EIB. Similarly, human beta-defensin 2 was modestly increased in runners who developed bronchoconstriction. Notably, S100A8 levels correlated negatively with lung function parameters, including forced expiratory volume and mid-expiratory flows. These findings suggest that endurance running induces systemic inflammatory responses and modulates innate immune peptides, particularly in individuals prone to bronchoconstriction. These peptides may serve as biomarkers of respiratory stress and help guide personalized strategies in endurance sports.

Proteins of the plakin family are predominantly expressed in the epidermis and play a crucial role in cytoskeletal assembly by crosslinking intracellular structural components with cell-cell junctions and the plasma membrane. While most plakins are critical for maintaining epidermal integrity, the role of epiplakin (EPPK1) in inflammatory skin disorders has not been thoroughly investigated. We therefore used single-cell RNA sequencing (scRNAseq) analysis, immunofluorescence, and ex vivo cytokine treatment of human skin explants to investigate EPPK1 regulation in psoriasis. ScRNAseq analysis of psoriatic and healthy skin revealed that EPPK1 was the only member of the plakin family showing specific downregulation in the epidermis of psoriatic lesions. This finding was corroborated at the protein level by immunostaining of human psoriasis samples showing a specific downregulation of EPPK1 in the suprabasal granular layer of psoriatic epidermis. Transcriptomic profiling of Eppk1-/- murine epidermis revealed reduced expression of genes involved in epithelial adhesion and lipid metabolism, partially overlapping with the psoriatic keratinocyte signature, suggesting that EPPK1 loss may predispose the skin to barrier dysfunction under inflammatory stress. Investigation of the mechanism underlying the EPPK1 regulation in psoriasis revealed that interferon-γ (IFN-γ) was the main cytokine involved in its downregulation in human ex vivo skin. Collectively, our findings demonstrate a specific IFN-γ-dependent downregulation of EPPK1 in psoriasis, suggesting that lack of EPPK1 might contribute to the epithelial defects observed in this inflammatory skin condition.

Intracellular signaling factors are important targets for immunosuppressive drugs. We report a selective induction of the serine/threonine kinase Provirus Integration site for Moloney murine leukemia virus 3 (PIM3), but not its homologs PIM1 and PIM2, in activated human T cells. Specific pharmacological inhibition and CRISPR/Cas9-mediated knockout of PIM3 in primary human T cells within 2D and 3D cell culture models uncovered essential roles of this kinase in regulating T cell proliferation, viability, migration, metabolic activity, and cytotoxicity. Furthermore, PIM3 inhibition resulted in immunosuppressive effects in human immune organoids. Mass spectrometry-based target protein identification coupled with genome engineering revealed that PIM3 targets the Nucleolar protein Interacting with the FHA domain of MKI67 (NIFK) to control T cell proliferation, establishing a novel regulatory circuit that could be targeted therapeutically in conditions involving T cell hyperproliferation. Elevated PIM3 expression was detected in pathologic T cell infiltrates in human patients and pre-clinical models. Together, these findings identify PIM3 as a promising new target for immunosuppressive therapies. Supported by the Austrian Science Funds (FWF) project P34728-B Immune Response Regulation: Molecular Mechanisms (IRM)

Hyaluronic acid was proposed to support soft tissue recession surgery and guided tissue regeneration. The molecular mechanisms through which hyaluronic acid modulates the response of connective tissue cells remain elusive. To elucidate the impact of hyaluronic acid on the connective tissue cells, we used bulk RNA sequencing to determine the changes in the genetic signature of gingival fibroblasts exposed to 1.6% cross-linked hyaluronic acid and 0.2% natural hyaluronic acid. Transcriptome-wide changes were modest. Even when implementing a minimum of 1.5 log2 fold-change and a significance threshold of 1.0 -log10, only a dozenth of genes were differentially expressed. Upregulated genes were PLK3, SLC16A6, IL6, HBEGF, DGKE, DUSP4, PTGS2, FOXC2, ATAD2B, NFATC2, and downregulated genes were MMP24 and PLXNA2. RT-PCR analysis supported the impact of hyaluronic acid on increasing the expression of a selected gene panel. The findings from bulk RNA sequencing suggest that gingival fibroblasts experience weak changes in their transcriptome when exposed to hyaluronic acid.

Objectives: During the last years, age recommendations for the use of biological prostheses rather than mechanical prostheses for surgical aortic valve replacement (sAVR) have been lowered considerably. We evaluated survival rates, major adverse cardiac events (MACEs), and reoperation risks after surgical (sM-AVR) and biological (sB-AVR) AVR, to provide data for the optimal prosthesis choice for middle aged patients between 50 and 65 years. Methods: We performed a population-based cohort study using Austrian Health System data from 2010-2020. Patients undergoing isolated sAVR (n=3761) were categorized into sM-AVR (n=1018) and sB-AVR (n=2743) groups. Propensity score matching (PSM) was applied to balance covariates. The primary endpoint was all-cause mortality. Secondary endpoints included MACEs, reoperation, stroke, bleeding, and survival post-reoperation. Outcomes were assessed using Cox regression and Kaplan-Meier analyses. Results: Patients undergoing sM-AVR had significantly lower all-cause mortality compared to sB-AVR (HR=1.352, p=0.003). sB-AVR was associated with higher risks of MACEs (HR=1.182, p=0.03) and reoperation (HR=2.338, p=0.002). Stroke and bleeding rates were comparable. All results were sustained after PSM. Conclusion: The findings highlight increased mortality, MACEs and reoperation risks associated with sB-AVR compared to sM-AVR. We observed superior long-term outcomes after sM-AVR, suggesting the need to reevaluate the expanding use of sB-AVR in younger patients. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of lower Austria (GS1-EK-4/722-2021) gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

View Large Image Figure ViewerDownload Hi-res image Download (PPT)Clinical Implications•Epidermolysis bullosa (EB)–derived skin fibroblasts show elevated dipeptidyl-peptidase 4 activity.•Sitagliptin restores the levels of fibronectin and fibril deposition in EB-derived fibroblasts.•Sitagliptin presents an interesting treatment strategy for skin fibrosis and scarring. •Epidermolysis bullosa (EB)–derived skin fibroblasts show elevated dipeptidyl-peptidase 4 activity.•Sitagliptin restores the levels of fibronectin and fibril deposition in EB-derived fibroblasts.•Sitagliptin presents an interesting treatment strategy for skin fibrosis and scarring. Researchers have long recognized dipeptidyl-peptidase 4 (DPP4) as a therapeutic target in diabetes mellitus attributable to its role in glucose metabolism. However, recent research has unveiled its multifaceted involvement in various physiological processes beyond glycemic control, particularly in fibrotic disorders (Figure 1). In their recent study, Zeyer et al., 2024Zeyer K.A. Bornert O. Nelea V. Bao X. Leytens A. Sharoyan S. et al.Dipeptidyl peptidase-4-mediated fibronectin processing evokes a profibrotic extracellular matrix [e-pub ahead of print].J Invest Dermatol. 2024; (accessed 27 May 2024)https://doi.org/10.1016/j.jid.2024.03.020Google Scholar shed light on the significance of DPP4 in skin fibrosis, particularly in the context of epidermolysis bullosa (EB), and the potential therapeutic implications of targeting DPP4 in mitigating fibrosis and scarring. This commentary discusses the broader implications of DPP4 inhibition in fibrosis and highlights future research and clinical application directions. DPP4, an S9 proline oligopeptidase family member, is a widespread serine protease cleaving N-terminal dipeptides adjacent to proline or alanine residues. When matrix metalloproteinases cleave DPP4 from the cell surfaces, it becomes soluble (soluble DPP4 [sDPP4]), retaining its catalytic and enzymatic capabilities. sDPP4 accounts for 95% of dipeptidyl peptidase activity in human serum. DPP4 is found in various organs and cell types, influencing T-cell functions such as proliferation, activation, and inflammation while also affecting wound healing by modulating macrophage recruitment and fibrosis. By cleaving N-terminal dipeptides, DPP4 deactivates active peptides and processes substrates, including chemokines and vasoactive peptides such as glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, and substance P. In addition, DPP4 binds to adenosine deaminase and extracellular matrix (ECM) components such as fibronectin (Ohm et al., 2023Ohm B. Moneke I. Jungraithmayr W. Targeting cluster of differentiation 26 / dipeptidyl peptidase 4 (CD26/DPP4) in organ fibrosis.Br J Pharmacol. 2023; 180: 2846-2861Google Scholar). The expression and function of DPP4 in the skin have been controversial. During embryonic and early postnatal development, researchers detected DPP4 in the papillary dermis of the mouse skin; however, in adult mice, DPP4 was predominantly expressed in the reticular dermis. In contrast to studies in mice, there are limited and conflicting data on the expression and function of DPP4 in human skin. Rinkevich et al., 2015Rinkevich Y. Walmsley G.G. Hu M.S. Maan Z.N. Newman A.M. Drukker M. et al.Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.Science. 2015; 348aaa2151Google Scholar were the first to describe a distinct fibroblast (FB) cell population with intrinsic fibrotic potential characterized by the expression of DPP4 (Rinkevich et al., 2015Rinkevich Y. Walmsley G.G. Hu M.S. Maan Z.N. Newman A.M. Drukker M. et al.Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.Science. 2015; 348aaa2151Google Scholar). Traditionally, DPP4 has been considered a marker of reticular FBs. However, recent single-cell sequencing studies revealed DPP4 expression in FBs of both dermal layers in human skin (Tabib et al., 2018Tabib T. Morse C. Wang T. Chen W. Lafyatis R. SFRP2/DPP4 and FMO1/LSP1 define major fibroblast populations in human skin.J Invest Dermatol. 2018; 138: 802-810Google Scholar; Vorstandlechner et al., 2020Vorstandlechner V. Laggner M. Kalinina P. Haslik W. Radtke C. Shaw L. et al.Deciphering the functional heterogeneity of skin fibroblasts using single-cell RNA sequencing.FASEB J. 2020; 34: 3677-3692Google Scholar). Tabib et al., 2018Tabib T. Morse C. Wang T. Chen W. Lafyatis R. SFRP2/DPP4 and FMO1/LSP1 define major fibroblast populations in human skin.J Invest Dermatol. 2018; 138: 802-810Google Scholar suggested a new major FB population characterized by DPP4 and SFRP2 expressions, whereas Korosec et al., 2019Korosec A. Frech S. Gesslbauer B. Vierhapper M. Radtke C. Petzelbauer P. et al.Lineage identity and location within the dermis determine the function of papillary and reticular fibroblasts in human skin.J Invest Dermatol. 2019; 139: 342-351Google Scholar found a broader distribution of DPP4+ FBs mainly in the reticular dermis but also in the papillary dermis. In 1995, researchers proposed DPP4 inhibition as a therapeutic approach for treating type 2 diabetes (Ohm et al., 2023Ohm B. Moneke I. Jungraithmayr W. Targeting cluster of differentiation 26 / dipeptidyl peptidase 4 (CD26/DPP4) in organ fibrosis.Br J Pharmacol. 2023; 180: 2846-2861Google Scholar). In the early 2000s, studies introduced gliptins as small molecules targeting DPP4, and the Food and Drugs Administration approved sitagliptin in 2006. Gliptins are highly specific pharmacological inhibitors of DPP4 activity that effectively control blood glucose levels by inhibiting the cleavage of glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide. As a result, they are now widely used as an oral medication for treating diabetes mellitus and have a favorable profile of good tolerability and low side effects (Pathak and Bridgeman, 2010Pathak R. Bridgeman M.B. Dipeptidyl peptidase-4 (DPP-4) inhibitors in the management of diabetes.P T. 2010; 35: 509-513Google Scholar). In addition, they do not cause hypoglycemic episodes and have very few drug interactions (Pathak and Bridgeman, 2010Pathak R. Bridgeman M.B. Dipeptidyl peptidase-4 (DPP-4) inhibitors in the management of diabetes.P T. 2010; 35: 509-513Google Scholar). Moreover, gliptins have shown antihypertensive, anti-inflammatory, antiapoptotic, and immunomodulatory effects independent of the incretin pathway (Pathak and Bridgeman, 2010Pathak R. Bridgeman M.B. Dipeptidyl peptidase-4 (DPP-4) inhibitors in the management of diabetes.P T. 2010; 35: 509-513Google Scholar). However, physicians increasingly use gliptins for their benefit in diabetes and various fibrotic diseases, including the kidney, heart, lung, and liver diseases (Ohm et al., 2023Ohm B. Moneke I. Jungraithmayr W. Targeting cluster of differentiation 26 / dipeptidyl peptidase 4 (CD26/DPP4) in organ fibrosis.Br J Pharmacol. 2023; 180: 2846-2861Google Scholar). Initially, anecdotal evidence showed that patients treated with gliptins experienced improved wound healing. Interestingly, scar formation was also significantly improved in these patients. Subsequently, retrospective studies showed a significant reduction in the risk of hypertrophic scars and keloid development in gliptin-treated patients undergoing sternotomy (Suwanai et al., 2020Suwanai H. Watanabe R. Sato M. Odawara M. Matsumura H. Dipeptidyl peptidase-4 inhibitor reduces the risk of developing hypertrophic scars and keloids following median sternotomy in diabetic patients: a nationwide retrospective cohort study using the National Database of Health Insurance Claims of Japan.Plast Reconstr Surg. 2020; 146: 83-89Google Scholar). In recent years, extensive research has focused on DPP4 and its interaction with TGFβ in fibrosis and scar formation. Upon TGFβ stimulation, FBs differentiate into myofibroblasts, characterized by alpha-smooth muscle actin expression. Myofibroblasts can also originate from various progenitor cells, including tissue-resident mesenchymal cells and inflammatory monocytes. TGFβ signaling and FB activation into myofibroblasts are crucial in all fibrotic conditions. However, systemic targeting of TGFβ has faced challenges attributable to its diverse effects, including autoimmune reactions and increased cancer risk (Ohm et al., 2023Ohm B. Moneke I. Jungraithmayr W. Targeting cluster of differentiation 26 / dipeptidyl peptidase 4 (CD26/DPP4) in organ fibrosis.Br J Pharmacol. 2023; 180: 2846-2861Google Scholar). Although TGFβ itself is not a direct DPP4 substrate, gliptins exhibit antifibrotic effects, possibly by modulating downstream interactions of DPP4 with TGFβ (Thielitz et al., 2008Thielitz A. Vetter R.W. Schultze B. Wrenger S. Simeoni L. Ansorge S. et al.Inhibitors of dipeptidyl peptidase IV-like activity mediate antifibrotic effects in normal and keloid-derived skin fibroblasts.J Invest Dermatol. 2008; 128: 855-866Google Scholar). Studies have shown that DPP4 inhibitors interfere with TGFβ signaling pathways and reduce TGFβ receptor expression. In addition, sDPP4 activates the NF-κB pathway (Ohm et al., 2023Ohm B. Moneke I. Jungraithmayr W. Targeting cluster of differentiation 26 / dipeptidyl peptidase 4 (CD26/DPP4) in organ fibrosis.Br J Pharmacol. 2023; 180: 2846-2861Google Scholar). Recent research has highlighted the role of DPP4 in hypertrophic scar formation in both humans and mice (Vorstandlechner et al., 2021Vorstandlechner V. Laggner M. Copic D. Klas K. Direder M. Chen Y. et al.The serine proteases dipeptidyl-peptidase 4 and urokinase are key molecules in human and mouse scar formation.Nat Commun. 2021; 12: 6242Google Scholar). In their recent study, Zeyer et al., 2024Zeyer K.A. Bornert O. Nelea V. Bao X. Leytens A. Sharoyan S. et al.Dipeptidyl peptidase-4-mediated fibronectin processing evokes a profibrotic extracellular matrix [e-pub ahead of print].J Invest Dermatol. 2024; (accessed 27 May 2024)https://doi.org/10.1016/j.jid.2024.03.020Google Scholar shed new light on the role of DPP4 in interacting with the ECM, with a particular focus on fibronectin, fibrillin, and microfibrils. They used a model of EB, a chronic inflammatory condition that leads to fibrosis, rather than traditional skin fibrotic entities such as skin scarring or scleroderma. Increased TGFβ activity and FB activation were found in EB-derived fibrosis, supporting its suitability for studying fibrotic mechanisms alongside chronic inflammation. Notably, DPP4 expression and activity were significantly increased in EB-derived FBs, further emphasizing its relevance in all fibrotic conditions. Interestingly, the researchers showed that DPP4 modulates ECM deposition through proteolysis of the fibronectin N-terminus and that treatment with sitagliptin led to the normalization of ECM deposition. The study by Zeyer et al., 2024Zeyer K.A. Bornert O. Nelea V. Bao X. Leytens A. Sharoyan S. et al.Dipeptidyl peptidase-4-mediated fibronectin processing evokes a profibrotic extracellular matrix [e-pub ahead of print].J Invest Dermatol. 2024; (accessed 27 May 2024)https://doi.org/10.1016/j.jid.2024.03.020Google Scholar certainly provides a basis for further interesting investigations. The researchers showed that inhibition of DPP4 attenuates the altered fibril deposition of EB-derived FBs and shotgun inhibition of proteases using aprotinin. Investigating different (serine) proteases would be worthwhile, particularly dipeptidyl-peptidase 7 and dipeptidyl-peptidase 8, which show a synergistic interaction with DPP4. In our work on serine proteases in hypertrophic scars, we have identified several other overexpressed serine proteases in human and mouse scars, including urokinase, serine protease 23 (PRSS23), HTRA1, and AEBP1 (Vorstandlechner et al., 2021Vorstandlechner V. Laggner M. Copic D. Klas K. Direder M. Chen Y. et al.The serine proteases dipeptidyl-peptidase 4 and urokinase are key molecules in human and mouse scar formation.Nat Commun. 2021; 12: 6242Google Scholar). It would be interesting to see whether these also interact with fibronectin or integrins to influence TGFβ activity. However, apart from urokinase, no commercially available pharmacological inhibitors for these serine proteases exist, which makes further investigation difficult. In addition, we wonder whether DPP4 binds to or cleaves other ECM components, as the researchers have shown with fibronectin. Indeed, DPP4 has been shown to form complexes with seprase (FB activation protein-α), thereby allowing the degradation of ECM proteins, further facilitating cell invasion to the site of injury (Ohm et al., 2023Ohm B. Moneke I. Jungraithmayr W. Targeting cluster of differentiation 26 / dipeptidyl peptidase 4 (CD26/DPP4) in organ fibrosis.Br J Pharmacol. 2023; 180: 2846-2861Google Scholar). We believe that separately testing the binding dynamics of DPP4 to, for example, collagens I and III, elastin, but also glycoproteins or matricellular components would provide valuable insights to better understand the potential cleavage products resulting from DPP4 interaction with ECM. Furthermore, it would be interesting to see whether the observed changes in fibronectin/fibril deposition and the corresponding rescue by DPP4 inhibitors would also hold for FBs derived from other fibrotic pathologies, such as scleroderma and renal, cardiac, and hepatic sclerosis. If this is not the scenario, it would suggest a distinct mechanism in EB, possibly involving a specific interplay between the chronic inflammatory environment and the absence of collagen VII. Finally, in every context of DPP4 and fibrosis, we have asked ourselves whether there are physiological regulators of DPP4 activity, such as endogenous DPP4 inhibitors. Future studies could evaluate whether some of the ECM/fibronectin or other DPP4 substrate cleavage products or plasma/cellular molecules exist that could inhibit DPP4 activity, thus allowing a negative feedback loop to control DPP4 activity. Despite the growing evidence and understanding of DPP4 in fibrosis, all current gliptin studies listed on ClinicalTrials.gov are in diabetes or fibrotic complications of diabetes, such as cystic fibrosis–related diabetes. We believe that the time is ripe to bring DPP4 into clinical use in nondiabetic patients with fibrosis, particularly skin fibrosis and scarring. In the next few years, we hope that a phase I topical safety and tolerability study of gliptins will be initiated, followed soon by phase II efficacy studies in skin fibrosis. Vera Vorstandlechner: http://orcid.org/0000-0002-5938-1552 Michael Mildner: http://orcid.org/0000-0002-6892-925X The authors state no conflict of interest. Dipeptidyl Peptidase-4–Mediated Fibronectin Processing Evokes a Profibrotic Extracellular MatrixJournal of Investigative DermatologyPreviewFibronectin serves as a platform to guide and facilitate deposition of collagen and fibrillin microfibrils. During development of fibrotic diseases, altered fibronectin deposition in the extracellular matrix (ECM) is generally an early event. After this, dysregulated organization of fibrillins and fibrillar collagens occurs. Because fibronectin is an essential orchestrator of healthy ECM, perturbation of its ECM-organizational capacity may be involved in development of fibrosis. To investigate this, we employed recessive dystrophic epidermolysis bullosa as a disease model with progressive, severe dermal fibrosis. Full-Text PDF Open Access