Imaging of metabolic activity adaptations to UV stress, drugs and differentiation at cellular resolution in skin and skin equivalents – Implications for oxidative UV damage

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The Secretome of Irradiated Peripheral Mononuclear Cells Attenuates Hypertrophic Skin Scarring

Hypertrophic scars can cause pain, movement restrictions, and reduction in the quality of life. Despite numerous options to treat hypertrophic scarring, efficient therapies are still scarce, and cellular mechanisms are not well understood. Factors secreted by peripheral blood mononuclear cells (PBMCsec) have been previously described for their beneficial effects on tissue regeneration. In this study, we investigated the effects of PBMCsec on skin scarring in mouse models and human scar explant cultures at single-cell resolution (scRNAseq). Mouse wounds and scars, and human mature scars were treated with PBMCsec intradermally and topically. The topical and intradermal application of PBMCsec regulated the expression of various genes involved in pro-fibrotic processes and tissue remodeling. We identified elastin as a common linchpin of anti-fibrotic action in both mouse and human scars. In vitro, we found that PBMCsec prevents TGFβ-mediated myofibroblast differentiation and attenuates abundant elastin expression with non-canonical signaling inhibition. Furthermore, the TGFβ-induced breakdown of elastic fibers was strongly inhibited by the addition of PBMCsec. In conclusion, we conducted an extensive study with multiple experimental approaches and ample scRNAseq data demonstrating the anti-fibrotic effect of PBMCsec on cutaneous scars in mouse and human experimental settings. These findings point at PBMCsec as a novel therapeutic option to treat skin scarring

Transglutaminase Activity Is Conserved in Stratified Epithelia and Skin Appendages of Mammals and Birds

The cross-linking of structural proteins is critical for establishing the mechanical stability of the epithelial compartments of the skin and skin appendages. The introduction of isopeptide bonds between glutamine and lysine residues depends on catalysis by transglutaminases and represents the main protein cross-linking mechanism besides the formation of disulfide bonds. Here, we used a fluorescent labeling protocol to localize the activity of transglutaminases on thin sections of the integument and its appendages in mammals and birds. In human tissues, transglutaminase activity was detected in the granular layer of the epidermis, suprabasal layers of the gingival epithelium, the duct of sweat glands, hair follicles and the nail matrix. In the skin appendages of chickens, transglutaminase activity was present in the claw matrix, the feather follicle sheath, the feather sheath and in differentiating keratinocytes of feather barb ridges. During chicken embryogenesis, active transglutaminase was found in the cornifying epidermis, the periderm and the subperiderm. Transglutaminase activity was also detected in the filiform papillae on the tongue of mice and in conical papillae on the tongue of chickens. In summary, our study reveals that transglutaminase activities are widely distributed in integumentary structures and suggests that transglutamination contributes to the cornification of hard skin appendages such as nails and feathers

Scientific Reports / Establishment of keratinocyte cell lines from human hair follicles

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.(VLID)471837

Secretome of peripheral blood mononuclear cells enhances wound healing.

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SEC(PBMC)). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SEC(PBMC) containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SEC(PBMC) on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SEC(PBMC) containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SEC(PBMC) induced proliferation and tube-formation in a matrigel-assay. In addition, SEC(PBMC) treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers

EGR1 Is Implicated in Right Ventricular Cardiac Remodeling Associated with Pulmonary Hypertension

Background: Pulmonary hypertension (PH) is a vasoconstrictive disease characterized by elevated mean pulmonary arterial pressure (mPAP) at rest. Idiopathic pulmonary arterial hypertension (iPAH) and chronic thromboembolic pulmonary hypertension (CTEPH) represent two distinct subtypes of PH. Persisting PH leads to right ventricular (RV) hypertrophy, heart failure, and death. RV performance predicts survival and surgical interventions re-establishing physiological mPAP reverse cardiac remodeling. Nonetheless, a considerable number of PH patients are deemed inoperable. The underlying mechanism(s) governing cardiac regeneration, however, remain largely elusive. Methods: In a longitudinal approach, we profiled the transcriptional landscapes of hypertrophic RVs and recovered hearts 3 months after surgery of iPAH and CTEPH patients. Results: Genes associated with cellular responses to inflammatory stimuli and metal ions were downregulated, and cardiac muscle tissue development was induced in iPAH after recovery. In CTEPH patients, genes related to muscle cell development were decreased, and genes governing cardiac conduction were upregulated in RVs following regeneration. Intriguingly, early growth response 1 (EGR1), a profibrotic regulator, was identified as a major transcription factor of hypertrophic RVs in iPAH and CTEPH. A histological assessment confirmed our biocomputational results, and suggested a pivotal role for EGR1 in RV vasculopathy. Conclusion: Our findings improved our understanding of the molecular events driving reverse cardiac remodeling following surgery. EGR1 might represent a promising candidate for targeted therapy of PH patients not eligible for surgical treatment

SEC^PBMC induces formation of new blood vessels.

<p>(<b>A</b>) Representative CD31 stainings of wounds underneath the original wound edge are shown. Scale bars: 50 µm (<b>B</b>) The numbers of CD31 positive cells underneath the newly formed epidermis were evaluated. The graph represents the mean of 15 animals in each group (*: p<0.01). (<b>C</b>) The area of the granulation tissue taken up by CD31⁺ cells was evaluated. The graph represents the mean of 15 animals in each group. (<b>D</b>) Cell cycle analysis of SEC^PBMC treated microvascular EC shows a strong increase in proliferating cells. VEGF treatment served as positive control. One representative experiment of three each done in triplicates is shown (*: p<0.01). (<b>E</b>) A tube formation assay is shown. Compared to medium alone SEC^PBMC strongly induced tube formation in a matrigel assay. One representative experiment of three is shown.</p

Apoptotic MNC-secretomes in experimental stroke

<p>Mixed Model Analysis (SAS output)</p> <p>The data were analyzed using linear mixed models for the neuroscore on treatment group and time-point with the factor animal included as a random effect. The MIXED procedure in SAS 9.3 was used to perform the calculations. The raw output contains information on the model specifications, the estimated error variance and random effects variance, the estimated regression coefficients, the covariance structure of the model coefficients and type III F-tests for the hypotheses of no effect of either fixed effect or their interactions. An interaction plot was drawn using the GLM procedure. This plot shows the individual observations and their sample mean values in each group and for each time-point. The group labels 0,1,2 and 3 in the raw output refer to the treatment group in setting 1, the control group in setting 1, the treatment group in setting 2 and the control group in setting 2, respectively.</p> <p>Original Western blots to Figure 5 </p> <p>Expression of proteins involved in cytoprotective pathways in human Astrocytes and Schwann Cells </p> <p>Astrocytes (page 1) or Schwann Cells (page 2) were stimulated with hMNCapo sec, control medium (served as control to treatment) or positive control (control to the measured protein). Original blots for all measured proteins are given in this raw data set (pages 1 and 2). For each blot, lanes (1), (2), and (3) correspond to the groups medium control [(1)=control to treatment], human apoptotic MNC-secretomes [(2)=treatment] and positive control [(3)=recombinant protein].<br>Bands in each blot are shown for phosphorylated CREB, total-CREB, phosphorylated Erk1/2, total-Erk 1/2, phosphorylated HSP27, total-HSP27, phosphorylated cJun, total-cJun, phosphorylated Akt, and total-Akt. The molecular weight (kDa) for each protein can be seen under each blot.<br>Ponceau staining was used as loading control for each group (1), (2), and (3) and suggest equal loading.</p> <p>Original Western blots to Figure 6 </p> <p>Expression of Phosphorylated CREB in Astrocytes and Neurons after stimulation with the active compound hMNCapo sec or control medium: </p> <p>Cultured human Astrocytes and Neurons were incubated with hMNCapo sec or control (cell culture-) medium at indicated concentrations. Original blots can be seen here.<br>Ponceau staining shows equal loading.</p> <p> </p

SEC^PBMC induces migration of human primary fibroblasts and keratinocytes.

<p>Scratch wounds of FB (<b>A</b>) and KC (<b>D</b>) are shown. One representative experiment of three each done in triplicates is shown. The mean-width of the gaps of nine scratch wounds after 18 h was measured and the percentage of closure for FB (<b>B</b>) and KC (<b>E</b>) was calculated. (*: p<0.01). Cells cycle analyses revealed no significant differences in FB (<b>C</b>) and in KC (<b>F</b>). One representative experiment of three each done in triplicates is shown. (<b>G</b>) Ki67 staining of medium and SEC^PBMC treated wounds showed no significant alterations in proliferating cells. Photographs were taken at the wound-edge of wounds 7 days after wounding. Ki67 staining is shown in red and nuclear staining is shown in blue. C  =  crust, E  =  newly formed epidermis, G  =  granulation tissue HF  =  hair follicle. One representative animal of 15 is shown. Scale bars: 50 µm. (<b>H</b>) The mean from 15 animals per group of Ki67 positive cells at the wound edge of one high power image was calculated.</p

List of Antibodies.

<p>Company addresses:</p>1<p>Bedford, MA, USA; ²Littleton, CO, USA; ³Cambridge, UK; ⁴NEB: New England Biolabs, Beverly, MA, USA; ⁵Turku Finland; ⁶Buckinghamshire, UK; ⁷Rockford, IL, USA; ⁸Eugene, OR, USA. n.d.: not done.</p