Response Of Dermal Fibroblasts Research Articles

A Novel Dehydrated Human Umbilical Cord Particulate Medical Device: Matrix Characterization, Performance, and Biocompatibility for the Management of Acute and Chronic Wounds.

Chronic wounds present a significant socioeconomic burden forecasted to increase in prevalence and cost. Minimally manipulated human placental tissues have been increasingly employed and proven to be advantageous in the treatment of chronic wounds, showing improved clinical outcomes and cost-effectiveness. However, technological advances have been constrained by minimal manipulation and homologous use criteria. This study focuses on the characterization of a novel dehydrated human umbilical cord particulate (dHUCP) medical device, which offers a unique allogeneic technological advancement and the first human birth tissue device for wound management. Characterization analyses illustrated a complex extracellular matrix composition conserved in the dHUCP device compared to native umbilical cord, with abundant collagens and glycosaminoglycans imbibing an intricate porous scaffold. Dermal fibroblasts readily attached to the intact scaffold of the dHUCP device. Furthermore, the dHUCP device elicited a significant paracrine proliferative response in dermal fibroblasts, in contrast to fibrillar collagen, a prevalent wound device. Biocompatibility testing in a porcine full-thickness wound model showed resorption of the dHUCP device and normal granulation tissue maturation during healing. The dHUCP device is a promising advancement in wound management biomaterials, offering a unique combination of structural complexity adept for challenging wound topographies and a microenvironment supportive of tissue regeneration.

Glucose-Induced Cellular Morphological Changes Across Mammalian Species

All organisms encounter environmental fluctuations and must contend with these changes to maintain homeostasis. Some mammals tolerate a wide range of intracellular conditions while others do not. Altered glucose levels are an example of a physiological challenge that can be encountered by cells. We studied responses of dermal fibroblasts obtained from skin biopsies to altered glucose treatments in culture using a comparative approach. Specifically, we investigated responses of mammals known to tolerate a range of blood glucose levels (fruit-eating geladas and Egyptian rousettes) as well as a hibernator (ground squirrel), which exhibits a marked winter decrease in glucose metabolism. As a comparison, we investigated species that maintain fairly stable blood glucose (human and rat). Fibroblasts were cultured at baseline (8mM glucose), then subjected to hypoglycemic (2.5mM) and hyperglycemic (30mM) treatment over 24h. We assayed glycolytic rate in human and rat fibroblasts using a Seahorse XFp analyzer, and found no significance between oxygen consumption or glycolysis across glucose treatments (all p>0.05). To investigate underlying cell phenotypes, we used Cell Painting, employing fluorescent stains to mark organelles. Hoechst 34580 stained DNA, to quantify number and size of nuclei, offering insights into proliferation. MitoTracker Deep Red visualized mitochondria, providing information about energy generation. Alexa Fluor 488 Concanavalin A conjugate marked endoplasmic reticulum. F-actin was stained with Alexa Fluor 555 Phalloidin conjugate, detecting cytoskeletal organization and overall cell structure. Images were analyzed with CellProfiler software, which extracts ~1400 morphological cell features, including area, compactness, fluorescence intensity, and cell connectivity to quantify subtle changes in phenotype. Each extracted feature will be compared across treatments and within a comparative context. We predict that fibroblasts exposed to hyperglycemia exhibit mitochondrial fragmentation and redistribution (MitoTracker) and increased production of reactive oxygen species (assessed with MitoSOX), which is linked to the opening of mitochondrial permeability transition pores. Cell Profiler analysis will identify structural changes that could indicate negative outcomes, such as mitophagy. Hyperglycemia may also induce endoplasmic stress, increasing lipid droplets and altering shape and size of the endoplasmic reticulum. In contrast, hypoglycemia will cause mitochondrial degradation. In both hyper- and hypoglycemia, proliferation rates are predicted to decrease, resulting in fewer nuclei and reduced cell connectivity. Phenotypic differences across species, evaluated in the context of their organismal phenotype, will help explain the mechanisms of cell function in mammals under environmental stress. Funding by NSF #2022046. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heat stress induced in vitro affects cell viability and gene expression of dermal fibroblasts from bovine and buffalo

Aim of study: To evaluate the response of dermal fibroblasts to heat stress and different time exposures on the cell survival and gene expression. Area of study: Belém city, Pará state. Brazil. Material and methods: Fibroblasts were isolated from ear skin of bovine (n= 4) and buffalo (n= 4), cultured in vitro until the 3rd passage and submitted to heat stress at 42°C for 3, 6 and 12 h, except for the negative control (38.5°C for 24 h). Cell survival was measured using Trypan blue, and RNA isolation was performed using Trizol method following qRT-PCR to quantify the relative expression of the inducible heat shock protein HSPA1A, the pro-apoptotic BAX and pro-inflammatory IFN-γ genes. Main results: Heat stress induced in vitro affected the cell viability and gene expression in a time-dependent manner. Gene expression was relatively lower in buffalo (p<0.05) than in bovine. Until 3 h of heat stress, HSPA1A showed a slight increase in both bovine and buffaloes, and BAX was 5.82-fold greater in bovine (p<0.05). After 6 h, HSPA1A was 75.81-fold (p<0.0001) and INF-γ was 20.15-fold greater (p<0.05) in bovine than buffalo. Only after 6 h the cell viability started to decrease significantly (p<0.05) in both species. Research highlights: Dermal fibroblasts of buffaloes and bovine were sensitive to heat stress induced in vitro, which was most detrimental to cell survival after 6 h. The expression of HSPA1A, BAX and INF-γ genes in response to heat stress indicate a slight sensibility of the dermal fibroblasts of bovine compared to their buffalo counterpart.

Modulatory Effect of Rosmarinic Acid on H2O2-Induced Adaptive Glycolytic Response in Dermal Fibroblasts.

Oxidative stress induces the adaptive response and alteration of energy metabolism across human cell types. Dermal fibroblasts shift their energy system to overload anaerobic glycolysis when exposed to sub-lethal hydrogen peroxide (H2O2). However, oxidative stress levels in the cells can be depleted by antioxidants, and such cellular changes can therefore be modulated. The present study aimed to investigate the modulatory effect of rosmarinic acid (a polyphenol antioxidant) against H2O2-induced reactive oxygen species (ROS) and the glycolytic adaptive response in fibroblasts. The results showed that H2O2 caused a significant ROS increase in the cells, and pre-treatment with rosmarinic acid (5-50 µM) decreased ROS significantly in the presence of glutathione. Rosmarinic acid modulated the adaptive response in H2O2-treated cells by decreasing glucose consumption and lactate production. The rosmarinic acid also recovered intracellular ATP and decreased NADPH production via the pentose phosphate pathway. Several glycolytic enzymes, including hexokinase-2 (HK-2), phosphofructokinase-2 (PFK-2), and lactate dehydrogenase A (LDHA), were downregulated in cells treated with rosmarinic acid. Furthermore, the key antioxidant enzymes: glutathione-disulfide reductase (GSR), glutathione peroxidase-1 (GPx-1), and peroxiredoxin-1 (Prx-1) and redox protein thioredoxin-1 (Trx-1) were upregulated in treated cells compared to control cells. To sum up, the rosmarinic acid could be used as an antioxidant against H2O2-induced adaptive responses in fibroblasts by modulating glucose metabolism, glycolytic genes, and GSH production. The present work indicates that rosmarinic acid holds promise in cell-based research applications for combating ROS and enhancing dermal fibroblast health.

Genetic and Functional Analyses of Cutibacterium Acnes Isolates Reveal the Association of a Linear Plasmid with Skin Inflammation

Cutibacterium acnes is a commensal bacterium on the skin that is generally well-tolerated, but different strain types have been hypothesized to contribute to the disease acne vulgaris. To understand how some strain types might contribute to skin inflammation, we generated a repository of C.acnes isolates from skin swabs of healthy subjects and subjects with acne and assessed their strain-level identity and capacity to stimulate cytokine release. Phylotype II K-type strains were more frequent on healthy and nonlesional skin of subjects with acne than those isolated from lesions. Phylotype IA-1 C-type strains were increased on lesional skin compared with those on healthy skin. The capacity to induce cytokines from cultured monocyte-derived dendritic cells was opposite to this action on sebocytes and keratinocytes and did not correlate with the strain types associated with the disease. Whole-genome sequencing revealed a linear plasmid in high-inflammatory isolates within similar strain types that had different proinflammatory responses. Single-cell RNA sequencing of mouse skin after intradermal injection showed that strains containing this plasmid induced a higher inflammatory response in dermal fibroblasts. These findings revealed that C.acnes strain type is insufficient to predict inflammation and that carriage of a plasmid could contribute to disease.

Transcriptome analysis of the effects of polarized photobiomodulation on human dermal fibroblasts.

Photobiomodulation (PBM), the therapeutic use of light, is used to treat a myriad of conditions, including the management of acute and chronic wounds. Despite the presence of clinical evidence surrounding PBM, the fundamental mechanisms underpinning its efficacy remain unclear. There are several properties of light that can be altered in the application of PBM, of these, polarization-the filtering of light into specified plane(s)-is an attractive variable to investigate. To evaluate transcriptomic changes in human dermal fibroblasts in response to polarized PBM. A total of 71 Differentially Expressed Genes (DEGs) are described. All DEGs were found in the polarized PBM group (P-PBM), relative to the control group (PC). Of the 71 DEGs, 10 genes were upregulated and 61 were downregulated. Most DEGs were either mitochondrial or extracellular matrix (ECM)-related. Gene Ontology (GO) analysis was then performed using the DEGs from the P-PBM vs. PC group. Within biological processes there were 95 terms found (p<0.05); in the molecular function there were 18 terms found (p<0.05); while in the cellular component there were 32 terms enriched (p<0.05). A KEGG pathways analysis was performed for the DEGs found in the P-PBM vs. PC group. This revealed 21 significantly enriched pathways (p<0.05). Finally, there were 24 significantly enriched reactome pathways when comparing the DEGs of the P-PBM vs. PC groups (p<0.05). The P-PBM DEGs were almost always down regulated compared to the comparator groups. This may be explained by the P-PBM treatment conditions decreasing the amount of cellular stress, hence causing a decreased mitochondria and ECM protective response. Alternatively, it could point to an alternate mechanism, outside the mitochondria, by which PBM exerts its effects. Additionally, PBM appears to have a more widespread effect on the mitochondria than previously thought, opening up many new avenues of investigation in the process.

IRAK4 inhibition dampens pathogenic processes driving inflammatory skin diseases.

Innate immunity not only shapes the way epithelial barriers interpret environmental cues but also drives adaptive responses. Therefore, modulators of innate immune responses are expected to have high therapeutic potential across immune-mediated inflammatory diseases. IRAK4 is a kinase that integrates signaling downstream of receptors acting at the interface between innate and adaptive immune responses, such as Toll-like receptors (TLRs), interleukin-1R (IL-1R), and IL-18R. Because effects of IRAK4 inhibition are stimulus, cell type, and species dependent, the evaluation of the therapeutic potential of IRAK4 inhibitors requires a highly translational approach. Here, we profiled a selective IRAK4 inhibitor, GLPG2534, in an extensive panel of models of inflammatory skin diseases, translationally expanding evidence from in vitro to in vivo and from mouse to human. In vitro, IRAK4 inhibition resulted in substantial inhibition of TLR and IL-1 responses in dendritic cells, keratinocytes, granulocytes, and T cells but only weakly affected dermal fibroblast responses. Furthermore, disease activity in murine models of skin inflammation (IL-23-, IL-33-, imiquimod-, and MC903-induced) was markedly dampened by IRAK4 inhibition. Last, inhibiting IRAK4 reversed pathogenic molecular signatures in human lesional psoriasis and atopic dermatitis biopsies. Over the variety of models used, IRAK4 inhibition consistently affected central mediators of psoriasis (IL-17A) and atopic dermatitis (IL-4 and IL-13). Overall, our data highlight IRAK4 as a central player in skin inflammatory processes and demonstrate the potential of IRAK4 inhibition as a therapeutic strategy in chronic inflammatory skin diseases.

Discrepant Activation Pattern of Inflammation and Pyroptosis Induced in Dermal Fibroblasts in Response to Dengue Virus Serotypes 1 and 2 and Nonstructural Protein 1.

Four serotypes of dengue virus (DENV-1 to DENV-4) cause mild to severe disease in humans through infected mosquito bites. Dermal fibroblasts were found to be susceptible to DENV, and this may play a critical role in establishing the initial infection stage. However, the cellular response induced by the different DENV serotypes in dermal fibroblasts during the early stage of infection remains unclear. To determine this, normal human dermal fibroblast WS1 cells were infected with DENV-1 or DENV-2. Compared with the response elicited by DENV-1 infection, DENV-2 induced a stronger innate inflammatory response and cell death in the WS1 cells. However, DENV-1 activated a higher level of pyroptosis signaling than did DENV-2, which was associated with higher virion production. Caspase-1 inhibitor Ac-YVAD-cmk and imipramine, an antidepressant drug, reduced DENV-mediated caspase-1 and interleukin 1β (IL-β) cleavage in the pyroptosis pathway. Ac-YVAD-cmk and imipramine downregulated DENV virion production in WS1 cells. Furthermore, DENV-1 and DENV-2 NS1 proteins promoted diverse activation levels of cell death, inflammatory response, and activation of caspase-1 and IL-β in dermal fibroblasts at different time points. Collectively, these data suggest that DENV-1, DENV-2, and their nonstructural protein 1 (NS1) induce discrepant activation patterns of inflammation and pyroptosis in dermal fibroblasts. The pyroptosis caused by virus and NS1 may facilitate DENV replication in dermal fibroblasts. IMPORTANCE Skin fibroblasts are the primary cells of DENV infection through mosquito bites. Establishing a successful infection in dermal fibroblasts might be critical for dengue disease. However, the cellular response induced by DENV in dermal fibroblasts remains unclear. In this in vitro study, we found that DENV-2 and DENV-1 showed different time course patterns of virus replication and inflammation in dermal fibroblasts. We demonstrated that DENV-1 and DNEV-2 and their viral protein NS1 activate the cellular pyroptosis response to regulate virus replication in dermal fibroblasts. This finding suggests that pyroptosis activation in the DENV primary inoculation site plays a role in the establishment of a DENV infection.

Unaffected Li-Fraumeni Syndrome Carrier Parent Demonstrates Allele-Specific mRNA Stabilization of Wild-Type TP53 Compared to Affected Offspring

Li-Fraumeni Syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is inherited by offspring of a carrier parent. p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Unexpectedly, some mutant TP53 carriers remain unaffected, while their children develop cancer early in life. To begin unravelling this paradox, the response of dermal fibroblasts (dFb) isolated from a child with LFS was compared to those from her unaffected father after UV exposure. Phospho-Chk1[S345], a key activator of cell cycle arrest, was increased by UV induction in the LFS patient compared to their unaffected parent dFb. This result, along with previous findings of reduced CDKN1A/p21 UV induction in affected dFb, suggest that cell cycle dysregulation may contribute to cancer onset in the affected LFS subject but not the unaffected parent. Mutant p53 protein and its promoter binding affinity were also higher in dFb from the LFS patient compared to their unaffected parent. These results were as predicted based on decreased mutant TP53 allele-specific mRNA expression previously found in unaffected dFb. Investigation of the potential mechanism regulating this TP53 allele-specific expression found that, while epigenetic promoter methylation was not detectable, TP53 wild-type mRNA was specifically stabilized in the unaffected dFb. Hence, the allele-specific stabilization of wild-type TP53 mRNA may allow an unaffected parent to counteract genotoxic stress by means more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS.

Functional responses of dermal fibroblasts to low nutrition and pro-inflammatory stimuli mimicking a wound environment in vitro.

Dermal fibroblasts (DF) constitute one of key cells involved in wound healing. However, the functions they perform in wound conditions remain poorly understood. This study involved exposing DF to low nutrition and to low nutrition + LPS for 5 d as conditions representing the wound. Although DF exhibited increasing metabolic activity in time under all conditions including control, the proliferation did not change in both low nutrition and low nutrition + LPS. Only the low nutrition + LPS was found to potentiate the migration and pro-inflammatory phenotype (IL6 release) of DF. The potential of DF to contract collagen hydrogel declined only under low nutrition as a consequence of low cell number. The expression of α-SMA was reduced under both conditions independently of the cell number. The remodeling capability of DF was affected under both conditions as documented by the enhanced MMP2 activity. Finally, the production of collagen type I was not affected by either condition. The study shows that low nutrition as the single factor is able to delay the healing process. Moreover, the addition of the mild pro-inflammatory stimulus represented by LPS may amplify the cell response in case of decreased α-SMA expression or excite DF to produce IL6 impairing the healing process.

Nicotinamide Mononucleotide and Coenzyme Q10 Protects Fibroblast Senescence Induced by Particulate Matter Preconditioned Mast Cells.

Particulate matter (PM) pollutants impose a certain degree of destruction and toxicity to the skin. Mast cells in the skin dermis could be activated by PMs that diffuse across the blood vessel after being inhaled. Mast cell degranulation in the dermis provides a kind of inflammatory insult to local fibroblasts. In this study, we evaluated human dermal fibroblast responses to conditioned medium from KU812 cells primed with PM. We found that PM promoted the production of proinflammatory cytokines in mast cells and that the cell secretome induced reactive oxygen species and mitochondrial reactive oxygen species production in dermal fibroblasts. Nicotinamide mononucleotide or coenzyme Q10 alleviated the generation of excessive ROS and mitochondrial ROS induced by the conditioned medium from PM-activated KU812 cells. PM-conditioned medium treatment increased the NF-κB expression in dermal fibroblasts, whereas NMN or Q10 inhibited p65 upregulation by PM. The reduced sirtuin 1 (SIRT 1) and nuclear factor erythroid 2-related Factor 2 (Nrf2) expression induced by PM-conditioned medium was reversed by NMN or Q10 in HDFs. Moreover, NMN or Q10 attenuated the expression of senescent β-galactosidase induced by PM-conditioned KU812 cell medium. These findings suggest that NMN or Q10 ameliorates PM-induced inflammation by improving the cellular oxidative status, suppressing proinflammatory NF-κB, and promoting the levels of the antioxidant and anti-inflammatory regulators Nrf2 and SIRT1 in HDFs. The present observations help to understand the factors that affect HDFs in the dermal microenvironment and the therapeutic role of NMN and Q10 as suppressors of skin aging.

The neuropeptide Substance P facilitates the transition from an inflammatory to proliferation phase-associated responses in dermal fibroblasts.

The wound healing process is a product of three successive and overlapping phases of inflammation, proliferation and remodelling. Considerable efforts have been invested in deconstructing the intercellular crosstalk that orchestrates tissue repair, and we investigated the role of neuropeptides released from peripheral neurons upon injury in mediating these interactions. Amongst the most abundant of these neuropeptides secreted by nerves in the skin, is Substance P (SP). Given the role of dermal fibroblasts in coordinating multiple processes in the wound healing program, the effect of SP on human dermal fibroblasts of different ages was evaluated. The use of a substrate that recapitulates the mechanical properties of the in vivo tissue revealed novel effects of SP on dermal fibroblasts, including a block in inflammatory cytokine expression. Moreover, SP can promote expression of some extracellular matrix components and generates signals that regulate angiogenesis. Interestingly, the response of fibroblasts to SP was reduced concomitant with donor age. Altogether, SP acts to inhibit the inflammatory responses and promote proliferation-associated responses in an age-dependent manner in dermal fibroblasts, suggesting a role as a molecular switch between the inflammatory and proliferative phases of the wound healing response.

Anti-Aging β-Klotho Gene-Activated Scaffold Promotes Rejuvenative Wound Healing Response in Human Adipose-Derived Stem Cells.

Wound healing requires a tight orchestration of complex cellular events. Disruption in the cell-signaling events can severely impair healing. The application of biomaterial scaffolds has shown healing potential; however, the potential is insufficient for optimal wound maturation. This study explored the functional impact of a collagen-chondroitin sulfate scaffold functionalized with nanoparticles carrying an anti-aging gene β-Klotho on human adipose-derived stem cells (ADSCs) for rejuvenative healing applications. We studied the response in the ADSCs in three phases: (1) transcriptional activities of pluripotency factors (Oct-4, Nanog and Sox-2), proliferation marker (Ki-67), wound healing regulators (TGF-β3 and TGF-β1); (2) paracrine bioactivity of the secretome generated by the ADSCs; and (3) regeneration of basement membrane (fibronectin, laminin, and collagen IV proteins) and expression of scar-associated proteins (α-SMA and elastin proteins) towards maturation. Overall, we found that the β-Klotho gene-activated scaffold offers controlled activation of ADSCs’ regenerative abilities. On day 3, the ADSCs on the gene-activated scaffold showed enhanced (2.5-fold) activation of transcription factor Oct-4 that was regulated transiently. This response was accompanied by a 3.6-fold increase in the expression of the anti-fibrotic gene TGF-β3. Through paracrine signaling, the ADSCs-laden gene-activated scaffold also controlled human endothelial angiogenesis and pro-fibrotic response in dermal fibroblasts. Towards maturation, the ADSCs-laden gene-activated scaffold further showed an enhanced regeneration of the basement membrane through increases in laminin (2.1-fold) and collagen IV (8.8-fold) deposition. The ADSCs also expressed 2-fold lower amounts of the scar-associated α-SMA protein with improved qualitative elastin matrix deposition. Collectively, we determined that the β-Klotho gene-activated scaffold possesses tremendous potential for wound healing and could advance stem cell-based therapy for rejuvenative healing applications.

The immune response in bovine primary dermal fibroblasts is influenced by Interleukin 8 promoter haplotype and vitamin D

Interleukin-8 (IL-8) is a potent inflammatory chemokine, and two gene promoter haplotypes have been previously reported to segregate in cattle populations. Our earlier work showed how these divergent IL8 genotypes influence IL-8 expression and other immune response parameters at a systemic level. Here we extend that work to characterise the influence of haplotype on the local immune response – in primary bovine dermal fibroblasts. Furthermore, we also investigated how this response is modulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Significant induction of IL8 expression was observed in cells from both haplotypes at 3 and 24 h post-stimulation with the TLR1/2 ligand, Pam3CSK4 and with the TLR4 ligand, LPS. IL8 expression was elevated in response to both LPS and Pam3CSK4 in fibroblasts carrying the IL8-h1 haplotype and this result was supported by significantly enhanced IL-8 protein secretion. Gene expression profiles for other known fibroblast immune mediators (SAA3 and CCL20) did not show significant differences between haplotypes but NOS2 gene expression was significantly elevated in response to vitamin D, even above the level detected in response to both TLR ligands. In conclusion, this work has demonstrated that the IL-8 response of dermal fibroblasts is dependent on IL8 haplotype and that the immune response profile in these cells is significantly differentially regulated by 1,25(OH)2D3. Fibroblasts have important immune response capacity and their function in driving inflammatory responses (including iNOS) is underappreciated. Understanding the relationship between cattle genotype and immune function is critically important for uncovering sustainable solutions for animal disease.

Targeting the α7 nicotinic acetylcholine receptor-A novel road towards the future treatment of skin diseases.

Nicotinic acetylcholine receptors (nAChRs) are members of the superfamily of neurotransmitter-gated ion channels. The natural ligand for nAChRs is the endogenous neurotransmitter acetylcholine. Among the nAChRs is the α7nAChR. It is not only expressed by neural tissues but also in the skin. A number of different resident cutaneous cell types including epidermal keratinocytes, sebocytes and dermal fibroblasts express functional α7nAChR. Moreover, cells of the immune system such as lymphocytes, macrophages and monocytes, playing an important role in skin homeostasis, also express α7nAChR. Translational research focusing on the exploitation of the α7nAChR in dermatology has revealed that this neuroendocrine receptor could be promising target for the treatment of inflammatory skin diseases. For example, α7nAChR agonists can counteract transforming growth factor-β1-mediated responses in dermal fibroblasts, key effector cells in scleroderma. In accordance with this α7nAChR, agonists are effective in both inflammation and non-inflammation-driven models of experimentally induced skin fibrosis. Moreover, α7nAChR agonists can modulate expression of proinflammatory cytokines in epidermal keratinocytes that are crucially involved in the pathogenesis of psoriasis and other inflammatory skin diseases. Finally, the capability of α7nAChR agonists to suppress ultraviolet light A/B-induced responses, for example production of proinflammatory cytokines and oxidative stress, the latter crucially involved in dermal photoageing, points to a potential of such agents in the prevention of extrinsic skin ageing. Therefore, emphasis on translational research targeting the α7nAChR in skin may lead to the development of new treatment and prevention modalities against fibrosclerotic skin diseases, psoriasis vulgaris, atopic dermatitis, acne, photodermatoses and extrinsic skin ageing.

Pathogenic Activation of Mesenchymal Stem Cells Is Induced by the Disease Microenvironment in Systemic Sclerosis.

In systemic sclerosis (SSc), a persistent tissue repair process leads to progressive fibrosis of the skin and internal organs. The role of mesenchymal stem cells (MSCs), which characteristically initiate and regulate tissue repair, has not been fully evaluated. We undertook this study to investigate whether dividing metakaryotic MSCs are present in SSc skin and to examine whether exposure to the disease microenvironment activates MSCs and leads to transdifferentiation. Skin biopsy material from patients with recent-onset diffuse SSc was examined by collagenase spread of 1-mm-thick surface-parallel sections, in order to identify dividing metakaryotic stem cells in each tissue plane. Adipose-derived MSCs from healthy controls were treated with dermal blister fluid (BF) from patients with diffuse SSc and profiled by next-generation sequencing, or they were evaluated for phenotypic changes relevant to SSc. Differential responses of dermal fibroblasts were studied in parallel. MSC-like cells undergoing active metakaryotic division were identified in SSc sections (but not control sections) most prominently in the deep dermis and adjacent to damaged microvessels, in both clinically involved and uninvolved skin. Furthermore, exposure to SSc BF caused selective MSC activation, inducing a myofibroblast signature, while reducing signatures of vascular repair and adipogenesis and enhancing migration and contractility. Microenvironmental factors implicated in inducing transdifferentiation included the profibrotic transforming growth factor β, the presence of lactate, and mechanosensing, while the microenvironment Th2 cytokine, interleukin-31, enhanced osteogenic commitment (calcinosis). Dividing MSC-like cells are present in the SSc disease microenvironment where multiple factors, likely acting in concert, promote transdifferentiation and lead to a complex and resistant disease state.

Sildenafil Reduces Expression and Release of IL-6 and IL-8 Induced by Reactive Oxygen Species in Systemic Sclerosis Fibroblasts.

Oxidative stress linked to vascular damage plays an important role in the pathogenesis of systemic sclerosis (SSc). Indeed, vascular damage at nailfold capillaroscopy in patients with Raynaud’s Phenomenon (RP) is a major risk factor for the development of SSc together with the presence of specific autoantiobodies. Here, we investigated the effects of the phosphodiesterase type 5 inhibitor (PDE5i) sildenafil, currently used in the management of RP, in modulating the proinflammatory response of dermal fibroblasts to oxidative stress in vitro. Human fibroblasts isolated from SSc patients and healthy controls were exposed to exogenous reactive oxygen species (ROS) (100 µM H2O2), in the presence or absence of sildenafil (1 µM). Treatment with sildenafil significantly reduced dermal fibroblast gene expression and cellular release of IL-6, known to play a central role in the pathogenesis of tissue damage in SSc and IL-8, directly induced by ROS. This reduction was associated with suppression of STAT3-, ERK-, NF-κB-, and PKB/AKT-dependent pathways. Our findings support the notion that the employment of PDE5i in the management of RP may be explored for its efficacy in modulating the oxidative stress-induced proinflammatory activation of dermal fibroblasts in vivo and may ultimately aid in the prevention of tissue damage caused by SSc.

Genome-wide expression analysis of the heat stress response in dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu × taurine) cattle.

The present study sought to evaluate mRNA expression profiles in the cultured dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu, Tharparkar × taurine, Holstein Friesian) cattle in response to heat stress. Bioinformatics' analysis identified temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of stress responses, protein repair, metabolism, protein transport, cell division, and apoptosis. The present microarray platform contains 51,338 synthesized oligonucleotide probes corresponding to at least 36,713 unigenes. A total of 11,183 and 8126 transcripts were differentially expressed with a fold change of ≥ 2 in Tharparkar and Karan-Fries cattle, respectively. Randomly selected real-time validation showed 83.33% correlation with microarray data. Functional annotation and pathway study of the differentially expressed transcripts or genes (DEGs) reveal that upregulated genes significantly (P < 0.05) affect protein processing and NOD-like receptor pathways (NLRs), while downregulated genes were significantly (P < 0.05) found to be associated with cell cycle, metabolism, and protein transport. Gene expression changes include activation of heat shock factors (HSFs), increased expression of heat shock proteins (HSPs), and apoptosis, while decreasing protein synthesis and another metabolism. These findings provide insights into the underlying mechanism of the physiology of heat stress in Tharparkar and Karan-Fries cattle. Understanding the biology and mechanisms of heat stress is critical to developing approaches to ameliorate current production issues for improving animal performance and agriculture economics in tropical climatic conditions. In conclusion, the present study indicates that heat stress differentially affects the expression of the significant number of genes associated with stress response, metabolism, apoptosis, and protein transport in dermal fibroblasts of Tharparkar and Karan-Fries cattle.

960 Innate immune antimicrobial response by retinoic acid during reactive adipogenesis is dependent on HIF1α

Cutaneous bacterial infection by Gram-positive bacteria induces an essential innate immune response in dermal fibroblasts resulting in rapid differentiation of a subset of fibroblasts into adipocytes (reactive adipogenesis). Induction of adipogenesis is accompanied by a large yet transient increase in production of the antimicrobial peptide (AMP) cathelicidin (Camp) by preadipocytes during transition to mature adipocytes. Exposure to retinoic acid (RA) further enhanced Camp expression in 3T3-L1 preadipocytes (129-fold vs. control p < 0.0001) and in vivo in mouse skin (7-fold vs. control, p < 0.001). When RA was present during bacterial infection with S. aureus, Camp expression increased 19-fold, p < 0.001. To understand the mechanism by which RA regulates Camp we examined the role of Hypoxia-inducible factor-1α (HIF-1α). Previous studies have shown that retinoic RA enhances hif1a expression in several cell types, but a connection between RA, HIF-1α and AMPs had not previously been suspected. In concordance with our hypothesis, a HIF-1α transcriptional binding site is present in the Camp promoter, and RA-treated 3T3-L1 cells exhibited ∼2-fold higher hif1a mRNA expression compared to differentiated controls (p < 0.001). Immunostaining and Western blot also showed significantly higher levels of HIF-1α after RA treatment in vitro. In mice, RA treatment resulted in nearly a 2-fold increase in hif1a expression (p < 0.001), while a ∼3-fold, p < 0.0001 increase was seen in mice treated with RA during S. aureus infection. Pharmacological induction of HIF-1α by addition of the agonist (mimosine) in 3T3-L1 cells resulted in enhanced Camp expression comparable to RA (37- and 30- fold respectively vs. control, p < 0.0001) while cotreatment of RA and mimosine was synergistic (112-fold vs. control, p < 0.0001). HIF-1α inhibition by the inhibitor acriflavine abolished Camp expression following RA treatment. These findings link HIF-1α and RA in the capacity of dermal adipogenesis to fight infection.

Impact of Human Dermal Microvascular Endothelial Cells on Primary Dermal Fibroblasts in Response to Inflammatory Stress.

The aim of the present study was to evaluate the impact of the microenvironment produced by dermal microvascular endothelial cells, secondary to a pro-inflammatory challenge, on 2D culture models using dermal fibroblasts and in 3D reconstructed skin model using dermal fibroblasts and keratinocytes from healthy donors. We hypothesized that specific microvascular endothelial low grade inflammation could change fibroblasts phenotype and be involved in extracellular matrix (ECM) modification and skin alteration. Following IFNγ, TNFα, IL-1β pro-inflammatory stress on Human Dermal Endothelial Cells (HDMEC) we observed the increased release of Chemokine ligand 2 (CCL2), IL-6 and IL-8 but not VEGF-A in the conditioned medium (CM). The subsequent addition of this endothelial pro-inflammatory CM in dermal fibroblasts revealed an upregulation of IL6, IL8 and CCL2 but no NF-κB gene expression. The resulting ECM formation was impaired with a reduction of the collagen 1 network and a decrease in COL1A1 gene expression in 2D and 3D models. Collagen 1 and pro-LOX protein expression were significantly reduced confirming an impairment of the collagen network related to endothelial inflammation secretion. To conclude, this work showed that, without any immune cells, the endothelial secretion in response to a pro-inflammatory stress is able to activate the fibroblasts that will maintain the pro-inflammatory environment and exacerbate ECM degradation.