Caught in action: how MSCs modulate atherosclerotic plaque.

Atherosclerosis (AS) is a medical condition marked by the stiffening and constriction of the arteries. This is caused by the accumulation of plaque, a substance made up of fat, cholesterol, calcium, and other elements present in the blood. Over time, this plaque solidifies and constricts the arteries, restricting the circulation of oxygen-rich blood to the organs and other body parts. The onset and progression of AS involve a continuous inflammatory response, including the infiltration of inflammatory cells, foam cells derived from monocytes/macrophages, and inflammatory cytokines and chemokines. Mesenchymal stromal cells (MSCs), a type of multipotent stem cells originating from various body tissues, have recently been demonstrated to have a protective and regulatory role in diseases involving inflammation. Consequently, the transplantation of MSCs is being proposed as a novel therapeutic strategy for atherosclerosis treatment. This mini-review intends to provide a summary of the regulatory effects of MSCs at the plaque site to lay the groundwork for therapeutic interventions.

Tissue Inhibitor of Metalloproteinases-1 Interacts with CD74 to Promote AKT Signaling, Monocyte Recruitment Responses, and Vascular Smooth Muscle Cell Proliferation.

Tissue inhibitor of metalloproteinases-1 (TIMP-1), an important regulator of matrix metalloproteinases (MMPs), has recently been shown to interact with CD74, a receptor for macrophage migration inhibitory factor (MIF). However, the biological effects mediated by TIMP-1 through CD74 remain largely unexplored. Using sequence alignment and in silico protein-protein docking analysis, we demonstrated that TIMP-1 shares residues with both MIF and MIF-2, crucial for CD74 binding, but not for CXCR4. Subcellular colocalization, immunoprecipitation, and internalization experiments supported these findings, demonstrating that TIMP-1 interacts with surface-expressed CD74, resulting in its internalization in a dose-dependent manner, as well as with a soluble CD74 ectodomain fragment (sCD74). This prompted us to study the effects of the TIMP-1-CD74 axis on monocytes and vascular smooth muscle cells (VSCMs) to assess its impact on vascular inflammation. A phospho-kinase array revealed the activation of serine/threonine kinases by TIMP-1 in THP-1 pre-monocytes, in particular AKT. Similarly, TIMP-1 dose-dependently triggered the phosphorylation of AKT and ERK1/2 in primary human monocytes. Importantly, Transwell migration, 3D-based Chemotaxis, and flow adhesion assays demonstrated that TIMP-1 engagement of CD74 strongly promotes the recruitment response of primary human monocytes, while live cell imaging studies revealed a profound activating effect on VSMC proliferation. Finally, re-analysis of scRNA-seq data highlighted the expression patterns of TIMP-1 and CD74 in human atherosclerotic lesions, thus, together with our experimental data, indicating a role for the TIMP-1-CD74 axis in vascular inflammation and atherosclerosis.

Properties and fate of human mesenchymal stem cells upon miRNA let-7f-promoted recruitment to atherosclerotic plaques.

AIMS: Atherosclerosis is a chronic inflammatory disease of the arteries leading to the formation of atheromatous plaques. Human mesenchymal stem cells (hMSCs) are recruited from the circulation into plaques where in response to their environment they adopt a phenotype with immunomodulatory properties. However, the mechanisms underlying hMSC function in these processes are unclear. Recently, we described that miRNA let-7f controls hMSC invasion guided by inflammatory cytokines and chemokines. Here, we investigated the role of let-7f in hMSC tropism to human atheromas and the effects of the plaque microenvironment on cell fate and release of soluble factors. METHODS AND RESULTS: Incubation of hMSCs with LL-37, an antimicrobial peptide abundantly found in plaques, increased biosynthesis of let-7f and N-formyl peptide receptor 2 (FPR2), enabling chemotactic invasion of the cells towards LL-37, as determined by qRT-PCR, flow cytometry, and cell invasion assay analysis. In an Apoe-/- mouse model of atherosclerosis, circulating hMSCs preferentially adhered to athero-prone endothelium. This property was facilitated by elevated levels of let-7f in the hMSCs, as assayed by ex vivo artery perfusion and two-photon laser scanning microscopy. Exposure of hMSCs to homogenized human atheromatous plaque material considerably induced the production of various cytokines, chemokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases, as studied by PCR array and western blot analysis. Moreover, exposure to human plaque extracts elicited differentiation of hMSCs into cells of the myogenic lineage, suggesting a potentially plaque-stabilizing effect. CONCLUSIONS: Our findings indicate that let-7f promotes hMSC tropism towards atheromas through the LL-37/FPR2 axis and demonstrate that hMSCs upon contact with human plaque environment develop a potentially athero-protective signature impacting the pathophysiology of atherosclerosis.

Let-7f miRNA regulates SDF-1α- and hypoxia-promoted migration of mesenchymal stem cells and attenuates mammary tumor growth upon exosomal release.

Bone marrow-derived human mesenchymal stem cells (hMSCs) are recruited to damaged or inflamed tissues where they contribute to tissue repair. This multi-step process involves chemokine-directed invasion of hMSCs and on-site release of factors that influence target cells or tumor tissues. However, the underlying molecular mechanisms are largely unclear. Previously, we described that microRNA let-7f controls hMSC differentiation. Here, we investigated the role of let-7f in chemotactic invasion and paracrine anti-tumor effects. Incubation with stromal cell-derived factor-1α (SDF-1α) or inflammatory cytokines upregulated let-7f expression in hMSCs. Transfection of hMSCs with let-7f mimics enhanced CXCR4-dependent invasion by augmentation of pericellular proteolysis and release of matrix metalloproteinase-9. Hypoxia-induced stabilization of the hypoxia-inducible factor 1 alpha in hMSCs promoted cell invasion via let-7f and activation of autophagy. Dependent on its endogenous level, let-7f facilitated hMSC motility and invasion through regulation of the autophagic flux in these cells. In addition, secreted let-7f encapsulated in exosomes was increased upon upregulation of endogenous let-7f by treatment of the cells with SDF-1α, hypoxia, or induction of autophagy. In recipient 4T1 tumor cells, hMSC-derived exosomal let-7f attenuated proliferation and invasion. Moreover, implantation of 3D spheroids composed of hMSCs and 4T1 cells into a breast cancer mouse model demonstrated that hMSCs overexpressing let-7f inhibited tumor growth in vivo. Our findings provide evidence that let-7f is pivotal in the regulation of hMSC invasion in response to inflammation and hypoxia, suggesting that exosomal let-7f exhibits paracrine anti-tumor effects.

Autophagy unleashes noncanonical microRNA functions.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which act by guiding AGO (argonaute) proteins to target RNA transcripts in the RNA-induced silencing complex (RISC). This macromolecular complex includes multiple additional components (e.g., TNRC6A) that allow for interaction with enzymes mediating inhibition of translation or RNA decay. However, miRNAs also reside in low-molecular weight complexes without being engaged in target repression, and their function in this context is largely unknown. Our recent findings show that endothelial cells exposed to protective high-shear stress or MTORC inhibition activate the macroautophagy/autophagy machinery to sustain viability by promoting differential trafficking of MIR126 strands and by enabling unconventional features of MIR126-5p. Whereas MIR126-3p is degraded upon autophagy activation, MIR126-5p interacts with the RNA-binding protein MEX3A to form a ternary complex with AGO2. This complex forms on the autophagosomal surface and facilitates its nuclear localization. Once in the nucleus, MIR126-5p dissociates from AGO2 and establishes aptamer-like interactions with the effector CASP3 (caspase 3). The binding to MIR126-5p prevents dimerization and proper active site formation of CASP3, thus inhibiting proteolytic activity and limiting apoptosis. Disrupting this pathway in vivo by genetic deletion of Mex3a or by specific deficiency of endothelial autophagy aggravates endothelial apoptosis and exacerbates the progression of atherosclerosis. The direct inhibition of CASP3 by MIR126-5p reveals a non-canonical mechanism by which miRNAs can modulate protein function and mediate the autophagy-apoptosis crosstalk.

Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/ß-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/ß-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Expression of surface-associated 82kDa-proMMP-9 in primary acute leukemia blast cells inversely correlates with patients' risk.

With its ability to degrade extracellular matrix proteins and activate growth factors and cytokines, matrix metalloproteinase (MMP)-9 is an important regulator of cell function. Previously, we reported that myeloid leukemic cells express a unique 82kDa-proMMP-9 variant on their cell surface that is not affected by its natural inhibitor. In this study, we generated monoclonal antibodies that specifically recognize 82kDa-proMMP-9. Flow cytometry analysis using these antibodies revealed significant surface expression of 82kDa-proMMP-9 in monocytes, but minimal amounts in T and B cells isolated from peripheral blood of nine healthy donors and 22 patients with acute myeloid leukemia (AML). In all AML patients, blasts expressed 82kDa-proMMP-9 at levels of 4%-46%, with significantly higher levels in patients with a better risk defined according to National Comprehensive Cancer Network (NCCN) guidelines (ρ = -0.748, p < 0.001) and favorable phenotype according to the French-American-British classification (p = 0.02) compared with patients with adverse prognoses. Receiver operating characteristic curve analysis confirmed the diagnostic accuracy of 82kDa-proMMP-9 measurement in AML blasts (area under the curve: 0.893 [0.739-1.000], p = 0.019). It led us to define a cutoff value of 11.5% for identifying patients with lower NCCN risk (p = 0.005) and with a tendency toward a higher probability of response to anthracycline-based therapy (p = 0.109) and increased event-free survival (p = 0.24). Thus, 82kDa-proMMP-9 expression on blasts may represent a novel independent marker of prognosis in patients with AML.

Small but smart: MicroRNAs orchestrate atherosclerosis development and progression.

MicroRNAs (miRNAs) are short non-coding RNA able to bind specific sequences on target messenger RNAs (mRNAs) and thereby to post-transcriptionally modulate gene expression. Being expressed in all vertebrate cell types, miRNAs have emerged as key players in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Over the past decade, knowledge concerning the contribution of miRNAs to human pathology has grown with an astonishing pace. In particular, a major involvement of miRNAs in atherosclerosis as a leading cause of global mortality has been supported by ample evidence from in vitro, in vivo and clinical studies. This review aims to summarize and highlight current concepts of miRNA function in the continuum of atherogenesis ranging from risk factors (i.e. dyslipidemia, diabetes, hypertension), to endothelial dysfunction up to the events leading to plaque rupture. Areas in need for further research and potential perspectives for translational applications will be scrutinized. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez.

Upregulation of miR-203 and miR-210 affect growth and differentiation of keratinocytes after exposure to sulfur mustard in normoxia and hypoxia.

Exposure of the skin to sulfur mustard (SM) results in long-term complications such as impaired tissue regeneration. Previous own studies in normal human epidermal keratinocytes (NHEK) treated with SM demonstrated reduced proliferation, premature differentiation and a restricted functionality of hypoxia-mediated signaling in the cells. Here, we investigated the involvement of microRNAs, miR-203 and miR-210, in these mechanisms. SM significantly upregulated the expression of miR-203 in NHEK when cultivated under normoxic and hypoxic conditions. SM had no effect on miR-210 under normoxia. However, miR-210 levels were greatly increased in NHEK when grown in hypoxia and further elevated upon exposure of the cells to SM. In normoxia and hypoxia, inhibition of miR-203 by transfection of NHEK with complementary oligonucleotides, anti-miR-203, attenuated the SM-induced impairment of metabolic activity and proliferation, and counteracted SM-promoted keratin-1 expression in these cells. Consistent ameliorating effects on dysregulated metabolic activity, proliferation and keratin-1 expression in SM-treated NHEK were obtained upon inhibition of miR-210 in these cells grown in hypoxia. Our findings provide evidence that miR-203 and miR-210 are key regulators in normal and SM-impaired keratinocyte functionality, and suggest potential usefulness of inhibitors against miR-203 and miR-210 for target-directed therapeutical intervention to improve re-epithelialization of SM-injured skin.

RECK (reversion-inducing cysteine-rich protein with Kazal motifs) regulates migration, differentiation and Wnt/ß-catenin signaling in human mesenchymal stem cells.

The membrane-anchored glycoprotein RECK (reversion-inducing cysteine-rich protein with Kazal motifs) inhibits expression and activity of certain matrix metalloproteinases (MMPs), thereby suppressing tumor cell metastasis. However, RECK's role in physiological cell function is largely unknown. Human mesenchymal stem cells (hMSCs) are able to differentiate into various cell types and represent promising tools in multiple clinical applications including the regeneration of injured tissues by endogenous or transplanted hMSCs. RNA interference of RECK in hMSCs revealed that endogenous RECK suppresses the transcription and biosynthesis of tissue inhibitor of metalloproteinases (TIMP)-2 but does not influence the expression of MMP-2, MMP-9, membrane type (MT)1-MMP and TIMP-1 in these cells. Knockdown of RECK in hMSCs promoted monolayer regeneration and chemotactic migration of hMSCs, as demonstrated by scratch wound and chemotaxis assay analyses. Moreover, expression of endogenous RECK was upregulated upon osteogenic differentiation and diminished after adipogenic differentiation of hMSCs. RECK depletion in hMSCs reduced their capacity to differentiate into the osteogenic lineage whereas adipogenesis was increased, demonstrating that RECK functions as a master switch between both pathways. Furthermore, knockdown of RECK in hMSCs attenuated the Wnt/ß-catenin signaling pathway as indicated by reduced stability and impaired transcriptional activity of ß-catenin. The latter was determined by analysis of the ß-catenin target genes Dickkopf1 (DKK1), axis inhibition protein 2 (AXIN2), runt-related transcription factor 2 (RUNX2) and a luciferase-based ß-catenin-activated reporter (BAR) assay. Our findings demonstrate that RECK is a regulator of hMSC functions suggesting that modulation of RECK may improve the development of hMSC-based therapeutical approaches in regenerative medicine.

Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor.

Skin exposure to sulfur mustard (SM) provokes long-term complications in wound healing. Similar to chronic wounds, SM-induced skin lesions are associated with low levels of oxygen in the wound tissue. Normally, skin cells respond to hypoxia by stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α modulates expression of genes including VEGFA, BNIP3, and MMP2 that control processes such as angiogenesis, growth, and extracellular proteolysis essential for proper wound healing. The results of our studies revealed that exposure of primary normal human epidermal keratinocytes (NHEK) and primary normal human dermal fibroblasts (NHDF) to SM significantly impaired hypoxia-induced HIF-1α stabilization and target gene expression in these cells. Addition of a selective inhibitor of the oxygen-sensitive prolyl hydroxylase domain-containing protein 2 (PHD-2), IOX2, fully recovered HIF-1α stability, nuclear translocation, and target gene expression in NHEK and NHDF. Moreover, functional studies using a scratch wound assay demonstrated that the application of IOX2 efficiently counteracted SM-mediated deficiencies in monolayer regeneration under hypoxic conditions in NHEK and NHDF. Our findings describe a pathomechanism by which SM negatively affects hypoxia-stimulated HIF-1α signaling in keratinocytes and fibroblasts and thus possibly contributes to delayed wound healing in SM-injured patients that could be treated with PHD-2 inhibitors.

MicroRNA-specific regulatory mechanisms in atherosclerosis.

During the past decade, the crucial role of microRNAs (miRs) controlling tissue homeostasis and disease in the cardiovascular system has become widely recognized. By controlling the expression levels of their targets, several miRs have been shown to modulate the function of endothelial cells, vascular smooth muscle cells, and macrophages, thereby regulating the development and progression of atherosclerosis. For instance, miR-155 can exacerbate early stages of atherosclerosis by increasing the inflammatory activation and disturbing efficient lipid handling in macrophages. Conversely, miRs can exert atheroprotective roles, as has been established for the complementary miR-126 strand pair, which forms a dual system sustaining the endothelial proliferative reserve and promoting endothelial regeneration to counteract atherogenic effects of disturbed flow and hyperlipidemia. Under some conditions, miRs are released from cells and are transported by microvesicles, ribonucleoprotein complexes, and lipoproteins, being remarkably stable in circulation. Conferred by such delivery modules, miRs can regulate target mRNAs in recipient cells, representing a new tool for cell-cell communication in the context of atherosclerotic disease. Here, we will discuss novel aspects of miR-mediated regulatory mechanisms, namely the regulation by competing RNA targets, miRNA tandems, or complementary miR strand pairs, as well as their potential diagnostic and therapeutic value in atherosclerosis. This article is part of a Special Issue entitled 'Non-coding RNAs'.

Wnt5a/ß-catenin signaling drives calcium-induced differentiation of human primary keratinocytes.

It is well established that a gradient of extracellular calcium within the epidermis regulates the differentiation of keratinocytes. However, the molecular mechanisms implicated in this process are not fully understood. RNA interference of the calcium-sensing receptor (CaSR) showed that CaSR is essential in calcium-induced differentiation of normal human epidermal keratinocytes (NHEKs) by increasing the levels of free intracellular calcium, which upregulates the expression of Wnt5a but not Wnt3a, Wnt4, and Dkk-1 in the cells. Subsequently, autocrine Wnt5a promotes the differentiation of NHEKs, determined by increased biosynthesis of keratin-1 and loricrin, whereas proliferation is suppressed. Addition of both Wnt5a and calcium to NHEKs activated the Wnt/ß-catenin signaling pathway as indicated by (i) increased stability of ß-catenin in the cells, (ii) enhanced ß-catenin transcriptional activity, demonstrated by a luciferase-based ß-catenin-activated reporter assay, and (iii) augmented Wnt/ß-catenin target gene expression. NHEKs depleted for ß-catenin had a significantly reduced susceptibility to calcium-induced differentiation. Knockdown of axin 2, an antagonist of ß-catenin stability, enhanced the biosynthesis of keratin-1 and loricrin in the cells. Our findings establish a directional crosstalk between CaSR and Wnt/ß-catenin signaling in keratinocyte differentiation via Wnt5a that acts as an autocrine stimulus in this process.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/ß-catenin signaling.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/ß-catenin signaling pathway as indicated by the increased stability and nuclear localization of ß-catenin in TIMP-1-deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced ß-catenin transcriptional activity, determined by Wnt/ß-catenin target gene expression analysis and a luciferase-based ß-catenin-activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on ß-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1-mediated effects on Wnt/ß-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of ß-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1-depleted hMSCs and demonstrably reduced axin 2, an antagonist of ß-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/ß-catenin activity.

Sulfur mustard induces differentiation in human primary keratinocytes: opposite roles of p38 and ERK1/2 MAPK.

The chemical warfare agent sulfur mustard (SM) severely affects the regeneration capacity of skin. The underlying molecular and cellular mechanisms, however, are far from clear. Here, we demonstrate that normal human epidermal keratinocytes (NHEK) after exposure to SM strongly upregulated expression of keratin-1, involucrin, and loricrin, thus indicating premature epidermal differentiation. Furthermore, proliferation was repressed after treatment with SM. Analysis of intracellular signaling in NHEK revealed that SM enhances phosphorylation, nuclear translocation, and activity of the mitogen-activated protein kinases (MAPK) p38 and ERK1/2. Inhibition of p38 activity downregulated expression of keratin-1 and loricrin, whereas blockage of ERK1/2 significantly stimulated biosynthesis of these markers, pointing to opposite roles of p38 and ERK1/2 in the differentiation process. Simultaneous interruption of p38 and ERK1/2 activity led to a decreased expression of keratin-1 and loricrin. This suggests that NHEK differentiation is essentially controlled by p38 activity which may be negatively influenced by ERK1/2 activity. Functional analysis demonstrated that SM affects NHEK in their ability to migrate through extracellular matrix which can be rescued upon application of an inhibitor of p38 activity. Thus, our findings indicate that SM triggers premature differentiation in keratinocytes via p38 activity which may contribute to impaired regeneration of SM-injured skin.

Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression.

Based on the capacity of mesenchymal stem cells (MSC) to differentiate into multiple cell types in vitro and in vivo, MCS may be a suitable source for cell therapy and regeneration strategies. A prerequisite for effective clinical applications of human MSC (hMSC) is a profound knowledge of signal transduction cascades that mediate processes like proliferation, targeted migration and differentiation. Recently, we identified the canonical Wnt signal transduction pathway as a key player in hMSC proliferation and invasion. To evaluate whether those findings are transferable to the equivalent counterparts in mice, we studied important steps in the wingless/int-1 (Wnt) signal transduction pathway in mouse MSC (mMSC) and mMSC carrying a T cell specific transcription factor (TCF)/lymphoid enhancer binding factor (LEF)-reporter transgene. We found that the induction of the canonical Wnt pathway resulted in the up-regulation of the known Wnt target gene cyclin D1, closely associated with an enhanced proliferation capacity of mMSC. Interestingly, the expression of the Wnt target gene membrane type 1-matrix metalloproteinase (MT1-MMP) was diminished in mMSC upon Wnt3a stimulation, which came along with an impaired invasion. In line with these findings, MMP-2 and MMP-9 expression levels in mMSC were also decreased after Wnt3a treatment. In contrast, inhibition of Wnt signalling by the knockdown of the transcriptional activator beta-catenin resulted in an up-regulation of MT1-MMP and mMSC invasion. By comparing these findings with the settings in hMSC, major differences in Wnt-regulated MMP expression were observed in mMSC. Thus, our data advice caution when mouse model systems represent the pre-clinical validation of MSC-mediated therapeutical approaches.

Matrix metalloproteinase-9 expression and release from skin fibroblasts interacting with keratinocytes: Upregulation in response to sulphur mustard.

Matrix metalloproteinases (MMPs), especially MMP-9 and MMP-2, degrade various proteins of the extracellular matrix, including collagen type IV the major component of basement membranes which also separate the epidermis from the dermis. Although previous work indicates the contribution of MMPs and their inhibitors (TIMPs) to the pathophysiology of skin lesions induced by the toxic chemical warefare agent sulphur mustard (SM), little is known about the underlying molecular and cellular mechanisms. In this study we demonstrate in a 3D-skin model that topical application of SM significantly upregulated basal MMP-9 mRNA expression and release from the cells as shown by qRT-PCR and zymography, whereas that of MMP-2, membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 remained almost unaffected by SM. Further studies in neonatal human dermal fibroblasts (NHDF) and HaCaT keratinocytes revealed that MMP-9 was not secreted from these cells, neither with or without exposure to SM. However, when NHDF and HaCaT were cocultivated, MMP-9 was expressed and released from the cell mixture, suggesting that interaction between both cell types is essential for MMP-9 production. Moreover, SM-treatment of NHDF/HaCaT cocultures further upregulated MMP-9 biosynthesis and secretion, which was consistent with our findings obtained in the 3D-skin model. Addition of conditioned medium derived from SM-exposed HaCaT cells to NHDF was able to stimulate MMP-9 secretion and also increased the migratory potential of NHDF as shown in a scratch-wound healing assay and a fluorescent cell invasion assay. In contrast, culture supernatants of SM-treated NHDF had not such an effect on HaCaT cells. Taken together, our findings provide first evidence that SM exposure of skin stimulates keratinocytes to release soluble factors which in turn induce enhanced MMP-9 secretion and invasiveness of fibroblasts in vitro. This provides a potential mechanism probably contributing to SM-evoked tissue injury in vivo.