MSCs rescue impaired wound healing in a murine LAD1 model by adaptive responses to low TGF-ß1 levels.

Mutations in the CD18 gene encoding the common ß-chain of ß2 integrins result in impaired wound healing in humans and mice suffering from leukocyte adhesion deficiency syndrome type 1 (LAD1). Transplantation of adipose tissue-derived mesenchymal stem cells (MSCs) restores normal healing of CD18-/- wounds by restoring the decreased TGF-ß1 concentrations. TGF-ß1 released from MSCs leads to enhanced myofibroblast differentiation, wound contraction, and vessel formation. We uncover that MSCs are equipped with a sensing mechanism for TGF-ß1 concentrations at wound sites. Low TGF-ß1 concentrations as occurring in CD18-/- wounds induce TGF-ß1 release from MSCs, whereas high TGF-ß1 concentrations suppress TGF-ß1 production. This regulation depends on TGF-ß receptor sensing and is relayed to microRNA-21 (miR-21), which subsequently suppresses the translation of Smad7, the negative regulator of TGF-ß1 signaling. Inactivation of TGF-ß receptor, or overexpression or silencing of miR-21 or Smad7, abrogates TGF-ß1 sensing, and thus prevents the adaptive MSC responses required for tissue repair.

TLR4-dependent shaping of the wound site by MSCs accelerates wound healing.

We here address the question whether the unique capacity of mesenchymal stem cells to re-establish tissue homeostasis depends on their potential to sense pathogen-associated molecular pattern and, in consequence, mount an adaptive response in the interest of tissue repair. After injection of MSCs primed with the bacterial wall component LPS into murine wounds, an unexpected acceleration of healing occurs, clearly exceeding that of non-primed MSCs. This correlates with a fundamental reprogramming of the transcriptome in LPS-treated MSCs as deduced from RNAseq analysis and its validation. A network of genes mediating the adaptive response through the Toll-like receptor 4 (TLR4) pathway responsible for neutrophil and macrophage recruitment and their activation profoundly contributes to enhanced wound healing. In fact, injection of LPS-primed MSCs silenced for TLR4 fails to accelerate wound healing. These unprecedented findings hold substantial promise to refine current MSC-based therapies for difficult-to-treat wounds.

Newly Defined ATP-Binding Cassette Subfamily B Member 5 Positive Dermal Mesenchymal Stem Cells Promote Healing of Chronic Iron-Overload Wounds via Secretion of Interleukin-1 Receptor Antagonist.

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP-binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+ -derived mesenchymal stem cells (MSCs) attenuated macrophage-dominated inflammation and thereby accelerated healing of full-thickness excisional wounds in the iron-overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin-1 receptor antagonist (IL-1RA) secreted by ABCB5+ -derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti-inflammatory M2 macrophages at the wound site. The beneficial anti-inflammatory effect of IL-1RA released from ABCB5+ -derived MSCs on human wound macrophages was conserved in humanized NOD-scid IL2rγ null mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker-enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans. Stem Cells 2019;37:1057-1074.

Alpha-Ketoglutarate Curbs Differentiation and Induces Cell Death in Mesenchymal Stromal Precursors with Mitochondrial Dysfunction.

Increased concentrations of reactive oxygen species (ROS) originating from dysfunctional mitochondria contribute to diverse aging-related degenerative disorders. But so far little is known about the impact of distinct ROS on metabolism and fate of stromal precursor cells. Here, we demonstrate that an increase in superoxide anion radicals due to superoxide dismutase 2 (Sod2) deficiency in stromal precursor cells suppress osteogenic and adipogenic differentiation through fundamental changes in the global metabolite landscape. Our data identify impairment of the pyruvate and l-glutamine metabolism causing toxic accumulation of alpha-ketoglutarate in the Sod2-deficient and intrinsically aged stromal precursor cells as a major cause for their reduced lineage differentiation. Alpha-ketoglutarate accumulation led to enhanced nucleocytoplasmic vacuolation and chromatin condensation-mediated cell death in Sod2-deficient stromal precursor cells as a consequence of DNA damage, Hif-1α instability, and reduced histone H3 (Lys27) acetylation. These findings hold promise for prevention and treatment of mitochondrial disorders commonly associated with aged individuals. Stem Cells 2017;35:1704-1718.

A model of the onset of the senescence associated secretory phenotype after DNA damage induced senescence.

Cells and tissues are exposed to stress from numerous sources. Senescence is a protective mechanism that prevents malignant tissue changes and constitutes a fundamental mechanism of aging. It can be accompanied by a senescence associated secretory phenotype (SASP) that causes chronic inflammation. We present a Boolean network model-based gene regulatory network of the SASP, incorporating published gene interaction data. The simulation results describe current biological knowledge. The model predicts different in-silico knockouts that prevent key SASP-mediators, IL-6 and IL-8, from getting activated upon DNA damage. The NF-κB Essential Modulator (NEMO) was the most promising in-silico knockout candidate and we were able to show its importance in the inhibition of IL-6 and IL-8 following DNA-damage in murine dermal fibroblasts in-vitro. We strengthen the speculated regulator function of the NF-κB signaling pathway in the onset and maintenance of the SASP using in-silico and in-vitro approaches. We were able to mechanistically show, that DNA damage mediated SASP triggering of IL-6 and IL-8 is mainly relayed through NF-κB, giving access to possible therapy targets for SASP-accompanied diseases.

Suppression of Neutrophil-Mediated Tissue Damage-A Novel Skill of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation. Stem Cells 2016;34:2393-2406.

UVA-1 exposure in vivo leads to an IL-6 surge within the skin.

UVA-1 is a known promotor of skin ageing. Cytokines like IL-1α, Il-1ß or TNF-α, VEGF and IL-6 orchestrate UV effects, and IL-6 is furthermore an effector of UVA-induced photoageing. We investigated how fractionated UVA-1 doses influence the cytokine milieu and especially the IL-6 levels in the skin in vivo. In a study with 35 participants, we exposed previously unirradiated human skin to three UVA-1 irradiation regimes. Cytokine levels in interstitial skin fluid were measured up to 48 hours postexposure and compared to unirradiated control skin fluid. Our results show that IL-6 levels increased significantly after UVA-1 exposure at selected time points. The other candidates IL-1α, Il-1ß or TNF-α and VEGF show no significant response after UVA-1 exposure in vivo. UVA-1 thus raises selectively IL-6 levels in vivo, a fact that underlines its role in photoageing and has potential implications for its modulatory effect on photoageing pathology.

Pathogenesis of wound healing disorders in the elderly.

The elderly constitute the age group most susceptible to wound healing disorders and chronic wounds, the most prevalent being venous leg ulcers, pressure ulcers, and diabetic foot ulcers. However, other age-associated diseases should also be taken into consideration in the diagnostic workup of chronic wounds, and not be underestimated. A better understanding of the pathomechanisms involved in the wound healing process is of key importance in combatting the difficulties associated with the treatment of chronic wounds. In recent decades, considerable progress has been made in the development of pioneering therapeutic strategies for chronic wounds. In this context, the use of growth factors and cytokines, tissue engineering, and cell therapy - including stem cells - have proven very promising. Nevertheless, prior to their introduction into routine clinical practice, large controlled clinical trials are required to assess the safety of these techniques.

Pathogenese von Wundheilungsstörungen bei älteren Patienten.

Ältere Menschen sind für Wundheilungsstörungen und chronische Wunden am anfälligsten, insbesondere für venöse Beinulzera, Dekubitalulzera und diabetische Fußulzera. Jedoch sollten bei der diagnostischen Abklärung chronischer Wunden weitere altersassoziierte Krankheiten in Betracht gezogen werden. Ein besseres Verständnis der an Wundheilungsstörungen beteiligten Pathomechanismen ist wichtig für die Bewältigung der Schwierigkeiten bei der Behandlung chronischer Wunden. In den letzten Jahrzehnten wurden beachtliche Fortschritte bei zukunftsweisenden Therapien chronischer Wunden erzielt. In diesem Zusammenhang haben sich Wachstumsfaktoren und Zytokine, Gewebeanzucht (Tissue Engineering) und Zelltherapie - auch mit Stammzellen - als sehr vielversprechend erwiesen. Vor Einführung in die klinische Praxis muss jedoch die Sicherheit dieser Techniken durch umfangreiche klinische Studien nachgewiesen werden.

Reduction of CD18 promotes expansion of inflammatory γδ T cells collaborating with CD4+ T cells in chronic murine psoriasiform dermatitis.

IL-17 is a critical factor in the pathogenesis of psoriasis and other inflammatory diseases. The impact of γδ T cells, accounting for an important source of IL-17 in acute murine IL-23- and imiquimod-induced skin inflammation, in human psoriasis is still unclear. Using the polygenic CD18(hypo) PL/J psoriasis mouse model spontaneously developing chronic psoriasiform dermatitis due to reduced CD18/ß2 integrin expression to 2-16% of wild-type levels, we investigated in this study the influence of adhesion molecule expression on generation of inflammatory γδ T cells and analyzed the occurrence of IL-17-producing γδ and CD4(+) T cells at different disease stages. Severity of CD18(hypo) PL/J psoriasiform dermatitis correlated with a loss of skin-resident Vγ5(+) T cells and concurrent skin infiltration with IL-17(+), IL-22(+), and TNF-α(+) γδTCR(low) cells preceded by increases in Vγ4(+) T cells in local lymph nodes. In vitro, reduced CD18 levels promoted expansion of inflammatory memory-type γδ T cells in response to IL-7. Similar to IL-17 or IL-23/p19 depletion, injection of diseased CD18(hypo) PL/J mice with anti-γδTCR Abs significantly reduced skin inflammation and largely eliminated pathological γδ and CD4(+) T cells. Moreover, CD18(hypo) γδ T cells induced allogeneic CD4(+) T cell responses more potently than CD18(wt) counterparts and, upon adoptive transfer, triggered psoriasiform dermatitis in susceptible hosts. These results demonstrate a novel function of reduced CD18 levels in generation of pathological γδ T cells that was confirmed by detection of increases in CD18(low) γδ T cells in psoriasis patients and may also have implications for other inflammatory diseases.

The skin of the naked mole-rat and its resilience against aging and cancer.

The naked mole-rat (NMR) Heterocephalus glaber (from the Greek/latin words ἕτερος, heteros = divergent, κεφαλή, kephale = head and glabra = hairless) was first described by Rüppell (Fig. 1) and belongs to the Hystricognath (from the Greek words ὕστριξ, hystrix = porcupine and γνάθος, gnathos = jaw) as a suborder of rodents. NMR are characterized by the highest longevity among rodents and reveal a profound cancer resistance. Details of its skin-specific protective and resistance mechanisms against aging and carcinogenesis have so far not been adequately characterized. Recently, our knowledge of NMR skin biology was complemented and expanded by published data using state-of-the art histological and molecular techniques. Here we review and integrate novel published data regarding skin morphology and histology of the aging NMR and the underlying mechanisms at the cellular and molecular level. We relate this data to the longevity of the NMR and its resistance to neoplastic transformation and discuss further open questions to understand its extraordinary longevity. In addition, we will address the exposome, defined as "the total of all non-genetic, endogenous and exogenous environmental influences" on the skin, respiratory tract, stomach, and intestine. Finally, we will discuss in perspective further intriguing possibilities arising from the interaction of skin with other organs.

[Skin aging-cellular senescence : What is the future?] / Hautalterung ­ zelluläre Seneszenz : Wohin geht die Reise?

BACKGROUND: Cellular senescence is the main cause of skin and organ aging and is associated with a wide range of aging-related diseases. OBJECTIVES: To understand which senolytics, senomorphics, and cell-based therapies have been developed to alleviate and even rejuvenate skin aging and reduce cellular senescence. METHODS: Basic literature for the mode of action of senolytics and senomorphics and their clinical perspectives in daily routine are discussed. RESULTS: Various causes lead to mitochondrial dysfunction and the activation of pro-aging signaling pathways, which eventually lead to cellular senescence with degradation of structural proteins of the dermal connective tissue and severe suppression of regenerative stem cell niches of the skin. CONCLUSIONS: Depletion of senescent cells suppress skin aging and enforce rejuvenation of skin and other organs and their function. The removal of senescent cells by cells of the native immune system is severely disturbed during aging. Selected senolytics and senomorphics are approved and are already on the market.

Angiogenin Released from ABCB5+ Stromal Precursors Improves Healing of Diabetic Wounds by Promoting Angiogenesis.

Severe angiopathy is a major driver for diabetes-associated secondary complications. Knowledge on the underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors at the edge of nonhealing diabetic wounds in a murine db/db model, closely mirroring human type 2 diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ stromal precursors. This compensates for the profoundly reduced angiogenin expression in nontreated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ stromal precursors before injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice, implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining stromal precursors-based therapies of nonhealing diabetic foot ulcers and other pathologies with impaired angiogenesis.

Artefactual effects of oxygen on cell culture models of cellular senescence and stem cell biology.

In life sciences, modelling of the in vivo conditions using in vitro models is an important tool to generate knowledge. Although aerobic organisms including mammals depend on accurate oxygen tension, mimicking physiological conditions in cell culture experiments is not very common. Due to the need for simple technical and experimental design, the requirement for simulating the in vivo oxygen tension parameters has been neglected over long time. Fortunately, due to increasing knowledge in recent years the attention has shifted towards this scientific demand. In this short review, we summarize data substantiating the necessity to adequately mimic physiological oxygen tension using cell culture models in life science research.

TGF-beta-dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis.

Dysfunctional Tregs have been identified in individuals with psoriasis. However, their role in the pathogenesis of the disease remains unclear. Here we explored the effect of diminished CD18 (beta2 integrin) expression on the function of CD4+CD25+CD127(-) Tregs using the Cd18 hypomorphic (Cd18hypo) PL/J mouse model of psoriasis that closely resembles the human disease. We found that reduced CD18 expression impaired cell-cell contact between Tregs and DCs. This led to dysfunctional Tregs, which both failed to suppress the pathogenic T cells and promoted the onset and severity of the disease. This failure was TGF-beta-dependent, as Tregs derived from Cd18hypo PL/J mice had diminished TGF-beta1 expression. Adoptive transfer of Tregs expressing wild-type levels of CD18 into affected Cd18hypo PL/J mice resulted in a substantial improvement of the psoriasiform skin disease, which did not occur upon coinjection of the cells with TGF-beta-specific neutralizing antibody. Our data indicate a primary dysfunction of Cd18hypo Tregs, allowing subsequent hyperproliferation of pathogenic T cells in the Cd18hypo PL/J mouse model of psoriasis. This study may provide a step forward in our understanding of the unique role of CD18 expression levels in avoiding autoimmunity.

Wound healing defect of Vav3-/- mice due to impaired {beta}2-integrin-dependent macrophage phagocytosis of apoptotic neutrophils.

Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for beta(2)-integrins-mediated macrophage functions during wound healing. Vav3(-/-) and Vav1(-/-)/Vav3(-/-) mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3(-/-) bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with beta(2)-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3(-/-) or beta(2)-integrin(CD18)(-/-) macrophages into wound margins failed to restore the healing defect of Vav3(-/-) mice, suggesting Vav3 to control the beta(2)-integrin-dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3(-/-) macrophages was causal for their reduced release of active transforming growth factor (TGF)-beta(1), for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-beta(1) deficiency in Vav3(-/-) macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-beta(1) into wounds of Vav3(-/-) mice fully rescued the delayed wound healing.

Connective Tissue and Fibroblast Senescence in Skin Aging.

There is increasing evidence that skin aging is significantly enforced by the accumulation of senescent dermal fibroblasts. Various stressors damaging macromolecules inside and outside fibroblasts are responsible. In addition, NK cells fail to adequately remove senescent (SEN) fibroblasts from tissues. SEN fibroblasts by the release of the proinflammatory, tissue degrading senescent-associated secretory phenotype factors and vesicles with distinct cargo impact on their endogenous niche and spread senescence and skin aging. In this review, we will further discuss less noticed facets, including the plasticity of distinct dermal fibroblast phenotypes, the underestimated impact of the extracellular matrix itself, and the depletion of fibroblast subsets on skin homeostasis and aging.

Persistent JunB activation in fibroblasts disrupts stem cell niche interactions enforcing skin aging.

Fibroblasts residing in the connective tissues constitute the stem cell niche, particularly in organs such as skin. Although the effect of fibroblasts on stem cell niches and organ aging is an emerging concept, the underlying mechanisms are largely unresolved. We report a mechanism of redox-dependent activation of transcription factor JunB, which, through concomitant upregulation of p16INK4A and repression of insulin growth factor-1 (IGF-1), initiates the installment of fibroblast senescence. Fibroblast senescence profoundly disrupts the metabolic and structural niche, and its essential interactions with different stem cells thus enforces depletion of stem cells pools and skin tissue decline. In fact, silencing of JunB in a fibroblast-niche-specific manner-by reinstatement of IGF-1 and p16 levels-restores skin stem cell pools and overall skin tissue integrity. Here, we report a role of JunB in the control of connective tissue niche and identified targets to combat skin aging and associated pathologies.

Slowly Repaired Bulky DNA Damages Modulate Cellular Redox Environment Leading to Premature Senescence.

Treatments on neoplastic diseases and cancer using genotoxic drugs often cause long-term health problems related to premature aging. The underlying mechanism is poorly understood. Based on the study of a long-lasting senescence-like growth arrest (10-12 weeks) of human dermal fibroblasts induced by psoralen plus UVA (PUVA) treatment, we here revealed that slowly repaired bulky DNA damages can serve as a "molecular scar" leading to reduced cell proliferation through persistent endogenous production of reactive oxygen species (ROS) that caused accelerated telomere erosion. The elevated levels of ROS were the results of mitochondrial dysfunction and the activation of NADPH oxidase (NOX). A combined inhibition of DNA-PK and PARP1 could suppress the level of ROS. Together with a reduced expression level of BRCA1 as well as the upregulation of PP2A and 53BP1, these data suggest that the NHEJ repair of DNA double-strand breaks may be the initial trigger of metabolic changes leading to ROS production. Further study showed that stimulation of the pentose phosphate pathway played an important role for NOX activation, and ROS could be efficiently suppressed by modulating the NADP/NADPH ratio. Interestingly, feeding cells with ribose-5-phosphate, a precursor for nucleotide biosynthesis that produced through the PPP, could evidently suppress the ROS level and prevent the cell enlargement related to mitochondrial biogenesis. Taken together, these results revealed an important signaling pathway between DNA damage repair and the cell metabolism, which contributed to the premature aging effects of PUVA, and may be generally applicable for a large category of chemotherapeutic reagents including many cancer drugs.

Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.

The CD18 hypomorphic (CD18hypo) PL/J mouse model clinically resembling human psoriasis is characterized by reduced expression of the common chain of beta2 integrins (CD11/CD18) to only 2-16% of WT levels. Previously we found that this chronic psoriasiform skin inflammation also depends on the presence of CD4+ T cells. Herein we investigated the role of macrophages in this CD18hypo mouse model. Activated macrophages were significantly increased in lesional skin as well as in inflamed skin draining lymph nodes (DLNs) of affected CD18hypo mice and were identified as being an important source of TNF-alpha in vivo. Both depletion of macrophages and neutralization of TNF-alpha resulted in a significant alleviation of psoriasiform skin inflammation. As monocyte chemotactic protein 1 was enhanced in lesional skin of affected CD18hypo mice, we intradermally injected recombinant murine monocyte chemotactic protein-1 (rJE/MCP-1) alone or in combination with rTNF-alpha into the skin of healthy CD18hypo mice. Only simultaneous injection of rJE/MCP-1 and rTNF-alpha, but neither substance alone, resulted in the induction of psoriasiform skin inflammation around the injection sites with recruitment and activation of macrophages. Collectively, our data suggest that maintenance of psoriasiform skin inflammation critically depends on efficient recruitment and activation of macrophages with sufficient release of TNF-alpha.