Fibroblasts Research Articles

Novel Gene Biomarkers Specific to Human Mesenchymal Stem Cells Isolated from Bone Marrow

In this paper, we present a comparative analysis of the transcriptomic profile of three different human cell types: hematopoietic stem cells (HSCs), bone marrow-derived mesenchymal stem cells (MSCs) and fibroblasts (FIBs). The work aims to identify unique genes that are differentially expressed as specific markers of bone marrow-derived MSCs, and to achieve this undertakes a detailed analysis of three independent datasets that include quantification of the global gene expression profiles of three primary cell types: HSCs, MSCs and FIBs. A robust bioinformatics method, called GlobalTest, is used to assess the specific association between one or more genes expressed in a sample and the outcome variable, that is, the ‘cell type’ provided as a single univariate response. This outcome variable is predicted for each sample tested, based on the expression profile of the specific genes that are used as input to the test. The precision of the tests is calculated along with the statistical sensitivity and specificity for each gene in each dataset, yielding four genes that mark MSCs with high accuracy. Among these, the best performer is the protein-coding gene Transgelin (TAGLN, Gene ID: 6876) (with a Positive Predictive Value > 0.96 and FDR < 0.001), which identifies MSCs better than any of the currently used standard markers: ENG (CD105), THY1 (CD90) or NT5E (CD73). The results are validated by RT-qPCR, providing novel gene biomarkers specific for human MSCs.

Efficient one-pot green synthesis of carboxymethyl cellulose/folic acid embedded ultrafine CeO2 nanocomposite and its superior multi-drug resistant antibacterial activity and anticancer activity.

Due to the prevalence of drug-resistant bacteria and the ongoing shortage of novel antibiotics as well as the challenge of treating breast cancer, the therapeutic and clinical sectors are consistently seeking effective nanomedicines. The incorporation of metal oxide nanoparticles with biological macromolecules and an organic compound emerges as a promising strategy to enhance breast cancer treatment and antibacterial activity against drug-resistant bacteria in various biomedical applications. This study aims to synthesize a unique nanocomposite consisting of CeO2 embedded with folic acid and carboxymethyl cellulose (CFC NC) via a green precipitation method using Moringa oleifera. Various spectroscopic and microscopic analyses are utilized to decipher the physicochemical characteristics of CFC NC and active phytocompounds of Moringa oleifera. Antibacterial study against MRSA (Methicillin-resistant Staphylococcus aureus) demonstrated a higher activity (95.6%) for CFC NC compared to its counterparts. The impact is attributed to reactive oxygen species (ROS), which induces a strong photo-oxidative stress, leading to the destruction of bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CFC NC are determined as 600µg/mL and 1000µg/mL, respectively. The anticancer activity against breast cancer cell resulted in the IC50 concentration of 10.8μg/mL and 8.2μg/mL for CeO2 and CFC NC respectively.The biocompatibility test was conducted against fibroblast cells and found 85% of the cells viable, with less toxicity. Therefore, the newly synthesized CFC NC has potential applications in healthcare and industry, enhancing human health conditions.

Formulation, Characterization and Evaluation of Minocycline Hydrochloride Loaded Polyurethane/Collagen Nanofibers via Electrospinning as Wound Dressings

Objective It was aimed to formulate minocyline.HCI loaded electrospun polyurethane/collagen (PU/Col) and polyurethane/collagen/polycaprolactone (PU/Col/PCL) nanofibers are intended for use as a wound dressing. Methods:The effect of polymer ratio and addition of PCL on the morphology, diameter, drug delivery, encapsulation efficiency, mechanical properties, antibacterial activity against Escherichia coli and Staphylococcus aureus, cytotoxicity, cell adhesion and proliferation were investigated. 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to test the cytotoxicity of the nanofibers on the human dermal fibroblast (HDF) cell line. Cell proliferation/adhesion was also determined by imaging HDF cells seeded on mats with scanning electron microscopy/fluorescence microscopy. Results:All nanofibers were bead-free and smooth in the diameter range of 866.7-882.4 nm. They had favorable encapsulation efficiency (≥79.3%), controlled drug release up to 24 hours and did not have cytotoxic effects. Although collagen was preferred for cell adhesion and proliferation, its spinnability and mechanical properties were poor. While PU improved the spinnability of collagen, its mechanical properties also enhanced with the addition of PCL. Nevertheles, all mats led to favorable cell adhesion and proliferation. All the nanofibers had antimicrobial activity against Escherichia coli and Staphylococcus aureus. Conclusion:In conclusion, PU/Col and PU/Col/PCL nanofiber mats, which had favorable encapsulation efficiency, controlled drug release and antibacterial activity at least 24 hours, cell viability, proliferation, adhesion, mechanical properties to be used as wound dressing, were successfully prepared.

Gene Delivery via Octadecylamine-Based Nanoparticles for iPSC Generation from CCD1072-SK Fibroblast Cells

This study presents a novel biotechnological approach using octadecylamine-based solid lipid nanoparticles (OCTNPs) for the first-time reprogramming of human CCD1072-SK fibroblast cells into induced pluripotent stem cells (iPSCs). OCTNPs, with an average size of 178.9 nm and a positive zeta potential of 22.8 mV, were synthesized, thoroughly characterized, and utilized as a non-viral vector to efficiently deliver reprogramming factors, achieving a remarkable transfection efficiency of 82.0%. iPSCs were characterized through immunofluorescence, flow cytometry, and RT-qPCR, confirming the expression of key pluripotency markers such as OCT4, SOX2, and KLF4, with alkaline phosphatase activity further validating their pluripotent state. Following this comprehensive characterization, the iPSCs were successfully differentiated into cardiomyocyte-like cells using 5-azacytidine. Our research highlights the innovative application of OCTNPs as a safe and effective alternative to viral vectors, addressing key limitations of iPSC reprogramming. The novel application of OCTNPs for efficient gene delivery demonstrates a powerful tool for advancing stem cell technologies, minimizing risks associated with viral vectors. These findings pave the way for further innovations in biotechnological applications, particularly in tissue engineering and personalized medicine.

Heterozygous de novo variants in HSPD1 cause hypomyelinating leukodystrophy through impaired HSP60 oligomerisation

IntroductionHypomyelinating leukodystrophies are a group of genetic disorders, characterised by severe permanent myelin deficiency. Their clinical features include developmental delay with or without neuroregression, nystagmus, central hypotonia, progressing to spasticity...

Catalytically Active Ti-Based Nanomaterials for Hydroxyl Radical Mediated Clinical X-Ray Enhancement.

Nanoparticle radioenhancement offers a promising strategy for augmenting radiotherapy by locally increasing radiation damage to tumor tissue. While past research has predominantly focused on nanomaterials with high atomic numbers, such as Au and HfO2, recent work has revealed that their radioenhancement efficacy decreases considerably when using clinically relevant megavoltage X-rays as opposed to the orthovoltage X-rays typically employed in research settings. Here, radiocatalytically active Ti-based nanomaterials for clinical X-ray therapy settings are designed. A range of candidate materials, including TiO2 (optionally decorated with Ag or Pt nanoseeds), Ti-containing metal-organic frameworks (MOFs), and 2D Ti-based carbides known as Ti3C2Tx MXenes, is investigated. It is demonstrated that these titanium-based candidates remain consistently performant across a wide energy spectrum, from orthovoltage to megavoltage. This sustained performance is attributed to the catalytic generation of reactive oxygen species, moving beyond the simple physical dose enhancements associated with photoelectric effects. Beyond titania, emergent materials like titanium-based MOFs and MXenes exhibit encouraging results, achieving dose-enhancement factors of up to three in human soft tissue sarcoma cells. Notably, these enhancements are absent in healthy human fibroblast cells under similar conditions of particle uptake, underscoring the selective impact of titanium-based materials in augmenting radiotherapy across the clinically relevant spectral range.

Antioxidant, Apoptotic, and Wound Healing Effects of Xantolis Cambodiana Extracts in Normal Human Dermal Fibroblasts.

Xantolis cambodiana has demonstrated significant antioxidant properties; however, the mechanisms underlying its protective effects against oxidative stress in cellular systems remain unexplored. This work investigated the efficacy of methanolic extracts in exhibiting oxidative damage and examined their mechanisms. The methanolic extract had a high phenolic content (116.89±29.01 mg GAE/g FW) and exhibited scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals with an IC50 value of 42.35±9.20 μg/ml. In addition, it had the highest antioxidant activity based on ferric-reducing antioxidant power (467.45±50.74 mg AA/100 g). Normal human dermal fibroblast (NHDF) cells were pretreated with the methanolic extract in a hydrogen peroxide (H2O2; 500 mM)-induced oxidative stress model, which resulted in a significant decrease in apoptosis and autophagy. Not only did the methanolic extract reduce mitochondrial membrane potential, it also stimulated NHDF cell migration and reduced reactive oxygen species production through mitochondrial dysfunction in the H2O2-induced stress model. These findings suggested that the methanolic extract (25 μg/ml) attenuated H2O2-induced oxidative stress in NHDF cells, significantly reducing apoptosis, autophagy, and mitochondrial dysfunction. Thus, this extract has the potential to support the wound healing process due to its antioxidant activity.

Jellein-I-conjugated Gold nanoparticles: Insights into the Antibacterial, Antibiofilm Activity against MRSA, and Anticancer Properties

Antimicrobial peptides (AMPs) and their conjugation with nanoparticles hold promise for targeting Methicillin-resistant Staphylococcus aureus (MRSA). In this study, Jellein-I peptide, with a cysteine at the C-terminus, was conjugated to gold nanoparticles (GNPs) to enhance its antibacterial, antibiofilm, and anticancer activities. The GNPs were synthesized and then conjugated to Jellein-I, forming Jellein-I-Cys-GNPs. The nanoparticles were characterized using UV-visible spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared (FTIR) spectroscopy. UV-visible spectroscopy, DLS, and TEM confirmed the formation of GNPs with an average size of 5.5 nm. The UV–visible spectroscopy and FTIR results indicated the presence of peptide functional groups on the surface of the GNPs. To assess antibacterial activity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed against MRSA strains. The conjugated Jellein-I exhibited lower MIC and MBC values (94 μM) compared to the free peptide (512 μM) against both clinical and standard MRSA strains. Additionally, GNP-conjugated Jellein-I demonstrated antibiofilm activity, inhibiting and removing biofilms while reducing metabolic activity by more than 82%, 44%, and 72%, respectively. Cell viability studies on rat dermal fibroblast (RDF) cells showed over 80% viability across all treatment concentrations. Furthermore, a clonogenic assay on the metastatic melanoma cell line (A375) demonstrated that GNP-conjugated Jellein-I had significantly better anticancer effects than the free peptide, even at a concentration as low as 9 μM. Our findings suggest that the conjugation of Jellein-I-Cys to GNPs could be a promising formulation for enhancing the antibacterial, antibiofilm, and anticancer activities of peptides. Further in vivo studies may establish this new nanoformulation as a potential therapeutic candidate for MRSA infections and cancer treatment.

Cytotoxic potential of Hemp seed oil and molecular docking studies with inflammatory markers of diabetic cardiomyopathy

Present study was to assess the potential cytotoxic effects of commercially available Hemp seed oil and the effectiveness of its compounds in inhibiting inflammatory markers associated with diabetic cardiomyopathy. The bioactive components of Hemp seed oil were analysed using gas chromatography coupled with mass spectrometry (GC–MS). To evaluate the oil's cytotoxic properties, a modified 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay was conducted on L929 cells (a mouse fibroblast cell line). Furthermore, an in vivo antiangiogenic assessment of the oil was performed using the chick chorioallantoic membrane (CAM) model, following a laboratory-standardized protocol. Docking and simulation studies were carried out to examine the binding and stability of the oil's compounds with inflammatory markers relevant to diabetic cardiomyopathy. The GC–MS analysis revealed that hemp seed oil contains several compounds, including Cannabidiol, Benzenemethanaminium, Dronabinol, Cannabinol, and Squalene. The cytotoxicity assessment on L929 cells demonstrated that Hemp seed oil did not exhibit any harmful effects at a concentration of 2.5 - 5 mg/mL. The CAM model confirmed the presence of antiangiogenic properties in Hemp seed oil, as evidenced by the inhibition of vascular patterns and blood vessel dissolution in the eggs. Additionally, docking and molecular dynamics (MD) simulations indicated that Benzenemethanaminium and Cannabielsoin exhibited the highest inhibitory efficacy and stability when interacting with IL-1β and C-reactive protein, respectively. This study suggests that Hemp seed oil possesses anti-inflammatory and anti-angiogenic properties, making it a potential herbal remedy for patients with diabetic cardiomyopathy.

Single-cell RNA sequencing analysis reveals the role of TXNDC5 in keloid formation

Objective: Thioredoxin domain-containing protein 5 (TXNDC5) is associated with fibrosis in a variety of organs, but its mechanism of action in keloid is unclear. In this study, we aimed to investigate the mechanism of TXNDC5 in keloid. Material and Methods: Single-cell RNA sequencing data of keloid and normal scar samples obtained from public databases were normalized and clustered using the Seurat package. Pathway enrich analysis was conducted using biological process enrichment analysis and Gene Set Enrichment Analysis (GSEA). In addition, TXNDC5 expression and its effects on migration and invasion of keloid fibroblasts (KFs) were validated based on cell function experiments. Results: A total of five cell types were obtained. The KF clusters were further clustered into two fibroblast subtypes (Fibroblast cells 1 and Fibroblast cells 2). Biological process enrichment analysis showed that transforming growth factor beta (TGF-β) signaling pathway was enriched in the two fibroblast subtypes. GSEA analysis demonstrated that genes in TGF-β signaling pathway were mainly enriched in Fibroblast cells 1, and that genes involved in cell proliferation, migration, and the TGF-β signaling pathway were all high-expressed in fibroblast cells 1. TXNDC5 was positively correlated with fibroblast proliferation, migration and TGF-β signaling pathway, and AUCell score. The cellular experiment confirmed that the messenger RNA and protein levels of TXNDC5 and TGF-β1 were high-expressed in KFs cells (P<0.001), and that knockdown of TXNDC5 downregulated TGF-β1 expression and inhibited migration and invasion of KFs (P<0.0001). Conclusion: Our study indicated that TGF-β signaling pathway was enriched in fibroblast cells, and TXNDC5 was positively correlated with proliferation, migration, and TGF-β signaling pathway. Cellular experiment demonstrated that knocking down TXNDC5 downregulated TGF-β1 expression, and suppressed migration and invasion of KFs. The current discoveries provided a new therapeutic strategy for the treatment of keloid.

HCMV strain- and cell type-specific alterations in membrane contact sites point to the convergent regulation of organelle remodeling.

Viruses are ubiquitous entities that infect organisms across the kingdoms of life. While viruses can infect a range of cells, tissues, and organisms, this aspect is often not explored in cell culture analyses. There is limited information about which infection-induced changes are shared or distinct in different cellular environments. The prevalent pathogen human cytomegalovirus (HCMV) remodels the structure and function of subcellular organelles and their interconnected networks formed by membrane contact sites (MCSs). A large portion of this knowledge has been derived from fibroblasts infected with a lab-adapted HCMV strain. Here, we assess strain- and cell type-specific alterations in MCSs and organelle remodeling induced by HCMV. Integrating quantitative mass spectrometry, super-resolution microscopy, and molecular virology assays, we compare infections with lab-adapted and low-passage HCMV strains in fibroblast and epithelial cells. We determine that, despite baseline proteome disparities between uninfected fibroblast and epithelial cells, infection induces convergent changes and is remarkably similar. We show that hallmarks of HCMV infection in fibroblasts, mitochondria-endoplasmic reticulum (ER) encapsulations and peroxisome proliferation, are also conserved in infected epithelial and macrophage-like cells. Exploring cell type-specific differences, we demonstrate that fibroblasts rely on endosomal cholesterol transport while epithelial cells rely on cholesterol from the Golgi. Despite these mechanistic differences, infections in both cell types result in phenotypically similar cholesterol accumulation at the viral assembly complex. Our findings highlight the adaptability of HCMV, in that infections can be tailored to the initial cell state by inducing both shared and unique proteome alterations, ultimately promoting a unified pro-viral environment.IMPORTANCEHuman cytomegalovirus (HCMV) establishes infections in diverse cell types throughout the body and is connected to a litany of diseases associated with each of these tissues. However, it is still not fully understood how HCMV replication varies in distinct cell types. Here, we compare HCMV replication with lab-adapted and low-passage strains in two primary sites of infection, lung fibroblasts and retinal epithelial cells. We discover that, despite displaying disparate protein compositions prior to infection, these cell types undergo convergent alterations upon HCMV infection, reaching a more similar cellular state late in infection. We find that remodeling of the subcellular landscape is a pervasive feature of HCMV infection, through alterations to both organelle structure-function and the interconnected networks they form via membrane contact sites. Our findings show how HCMV infection in different cell types induces both shared and divergent changes to cellular processes, ultimately leading to a more unified state.

Simple and Cost-Effective Generation of 3D Cell Sheets and Spheroids Using Curvature-Controlled Paraffin Wax Substrates

The challenges associated with animal testing in pharmaceutical development have driven the search for alternative in vitro models that mimic human tissues more accurately. In this study, we present a simple and cost-effective method for generating 3D cell sheets and spheroids using curvature-controlled paraffin wax films, which are easily accessible laboratory materials that eliminate the need for extracellular matrix (ECM) components or thermo-responsive polymers. By adjusting the curvature of the paraffin wax film, we successfully generated human periodontal ligament fibroblast (HPdLF) cell sheets and bone marrow-derived mesenchymal stem cell (hBMSC) spheroids. Key parameters, such as cell density, substrate curvature, and incubation time, were identified as critical factors for optimizing the formation of these 3D structures. In addition, the use of quantum dots (QDs) for cell tracking enabled long-term visualization and distinction between different cell types within complex tissue-like structures. We further demonstrated that wrapping the hBMSC spheroids with HPdLF cell sheets partially replicated the connective tissue structure of the periodontal ligament surrounding the tooth root. This highlights the potential of this platform for the construction of more sophisticated tissue-mimicking assemblies. In conclusion, curvature-controlled paraffin wax films provide a versatile and practical approach for 3D cell cultures. This simplifies the generation of both cell sheets and spheroids, offering a promising tool for tissue engineering and regenerative medicine applications, where precise cell-to-cell interactions are essential.Graphical abstract

Evaluation of Cytotoxic Activity of Cell Biomass from Eryngium planum and Lychnis flos-cuculi on Melanoma Cancer Cell

Melanoma is a malignant neoplasm of melanocytes in the skin, and its occurrence is increasing annually. Plant-based products contain active compounds with low toxicity and are accessible alternatives for melanoma cancer treatment. The biotechnology approach for obtaining plant-based products provides continuity and allows the high-yield production of phytochemically uniform biomass. The callus biomass of Eryngium planum L. and Lychnis flos-cuculi L. was induced on Murashige and Skoog (MS) medium supplemented with growth regulators. A combination of 3.0 mg/L of 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 0.3 mg/L of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea—(thidiazuron) was used to obtain E. planum callus. Meanwhile, the callus of L. flos-cuculi was cultivated on MS medium with 2.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D). Methanolic extracts (EpME and LFcME), including 40% MeOH fractions (Ep40MF and LFc40MF) and 80% MeOH fractions (Ep80MF and LFc80MF), of E. planum and L. flos-cuculi cell biomass were prepared. Their cytotoxicity activity was assessed in human fibroblast cells (MRC-5) and human melanoma cells (MeWo) by direct cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Qualitative analyses using thin-layer chromatography and UPLC-HRMS/MS chromatograms showed the presence of phenolic acids and saponins within the extracts and fractions of both cell biomasses. LFc80MF and Ep80MF showed the strongest toxicity against the MeWo cell line, with IC50 values of 47 ± 0.5 and 52 ± 4 μg/mL after 72 h of treatment. EpME and LFcME had IC50 values of 103 ± 4 and 147 ± 4 µg/mL, respectively. On the other hand, Ep40MF and LFc40MF were less toxic against the MeWo cell line compared to the extracts and 80% MeOH fractions, with IC50 values of 145 ± 10 and 172 ± 7 µg/mL. This study suggests that the obtained extracts and fractions of E. planum and L. flos-cuculi cell biomass potentially possess significant cytotoxic activity against MeWo cells, which work in a time and dose-dependent manner. Although the extracts and 80% MeOH fractions were more potent, the 40% MeOH was shown to be more selective against the MeWo than the control MRC-5 cells.

Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1

UVB radiation induces oxidative stress, DNA damage, and inflammation, leading to skin wrinkling, compromised barrier function, and an increased risk of carcinogenesis. Addressing or preventing photoaging may offer a promising therapeutic avenue for these conditions. Recent research indicated that mesenchymal stem cells (MSCs) exhibit significant therapeutic potential for various skin diseases. Given that extracellular vesicles (EV) can deliver diverse cargo to recipient cells and elicit similar therapeutic effects, we investigated the roles and underlying mechanisms of both adipose-derived MSC-derived EV (AMSC-EV) and umbilical cord-derived MSC-derived EV (HUMSC-EV) in photoaging. Our findings indicated that in vivo, treatment with AMSC-EV and HUMSC-EV resulted in improvements in wrinkles and skin hydration while also mitigating skin inflammation and thickness alterations in both the epidermis and dermis. Additionally, in vitro studies using human keratinocytes (HaCaTs), human dermal fibroblast cells (HDFs), and T-Skin models revealed that AMSC-EV and HUMSC-EV attenuated senescence, reduced levels of reactive oxygen species (ROS) and DNA damage, and alleviated inflammation induced by UVB. Furthermore, EV treatment enhanced cell viability and migration capacity in the epidermis and promoted extracellular matrix (ECM) remodeling in the dermis in photoaged cell models. Mechanistically, proteomics results showed that TIMP1 was highly expressed in both AMSC-EV and HUMSC-EV and could exert similar effects as MSC-EV. In addition, we found that EV and TIMP1 could inhibit Notch1 and downstream targets Hes1, P16, P21, and P53. Collectively, our data suggests that both AMSC-EV and HUMSC-EV attenuate skin photoaging through TIMP1/Notch1.

Topical Application of Melatonin in a Grapeseed Oil-based Microemulsion Accelerated Wound Healing in Rat Models

Objective: Melatonin has been associated with accelerated tissue regeneration and grapeseed oil has abundant unsaturated fatty acids, particularly linoleic acid that makes it a strong antioxidant, having the potential to promote wound healing by enhancing the presence of free radicals at the wound site. The study is aimed to evaluate the potential of a microemulsion gel using grapeseed oil as the organic phase and melatonin encapsulated in the vesicles to exhibit synergistic wound healing in Swiss albino rats. Materials and Method: Microemulsion containing grapeseed oil encapsulating melatonin was developed using the water-titration method. The surfactant and co-surfactant ratio (Smix) were fixed at 1:1. A pseudo-ternary diagram was used to determine the microemulsion zone and the developed microemulsion was further incorporated in carbopol 934P gel. The formulations were evaluated for their physicochemical properties and cytotoxicity assay. The optimized formulation was topically applied to cutaneous wounds of Swiss albino rat models. 30 Swiss albino rats were divided into five groups of 6 animals each: (i) Negative control group, (ii) Standard marketed formulation treated group, (iii) Optimized microemulsion containing Grapeseed oil and melatonin treated group, (iv) Grapeseed oil treated group and (v) Melatonin treated group. All the rats in each group were topically applied with the desired formulations daily for up to 14 days. Results: The treatment with a formulation comprising 10.18% Grapeseed oil, 24.88% water, and 64.94% Smix exhibited the highest entrapment efficiency of 86.65 ± 1.88% with an enhanced in vitro drug release of up to 83.02 ± 1.09%, also demonstrating first-order release kinetics. Furthermore, it did not inhibit L929 mouse fibroblast cell proliferation up to 500 μg/mL and promoted wound closure prior to other groups. Additionally, increased tissue maturation with higher collagen deposition was mostly seen by day 7. Thus demonstrating it is suitable for dermal application and sustained release of melatonin. The in vivo wound healing study and histological investigations on rat models demonstrated comparable results as observed in the marketed formulation of melatonin. Conclusion: The results showed that GSO oil based microemulsion encapsulating MEL could be a promising wound treatment option to exhibit accelerated wound healing effects.

Cannabidiol (CBD) modulates the transcriptional profile of ethanol-exposed human dermal fibroblast cells.

Cannabidiol (CBD) is abundant in the Cannabis sativa plant and exhibits complex immunomodulatory, anxiolytic, antioxidant, and antiepileptic properties. Several studies suggest that CBD could be used for different purposes in alcohol use disorder (AUD) and alcohol-related injuries to the brain and the liver. In this study, we focused on analyzing transcriptional alterations in human dermal fibroblasts (HDFs) cell line challenged simultaneously with ethanol and CBD as an ethanol-protective agent. We aimed to expose the genes and pathways responsible for at least some of the CBD effects in those cells that can be related to the AUD. Transcriptome analysis was performed using HDFs cell line that expresses both cannabinoid receptors and can metabolize ethanol through alcohol dehydrogenase activity. Fibroblasts are also responsible for the progression of liver fibrosis, a common comorbidity in AUD. With the use of a cellular test, we found that CBD at the lowest applied concentration (0.75μM) was able to stimulate depressed metabolism and reduce the level of apoptosis of cells treated with different concentrations of ethanol to the level observed in the control cells. Similar observations were made at the transcriptome level, in which cells treated with ethanol and CBD had similar expression profiles to the control cells. CBD also affects several genes connected with extracellular matrix formation (especially its collagen constituent), which can have potential implications for, e.g., fibrosis process.

Modulation of p300 acetylation to protect against doxorubicin-induced cardiotoxicity

Abstract Background Doxorubicin (DOX) is a widely-used anti-neoplastic agent, but the most common adverse reaction to its use is cardiotoxicity(1). DOX acts by producing reactive oxygen species (ROSs) that cause DNA damage(1,2), which activates various proteins, including the tumour suppressor p53 and the acetyltransferase p300. When activated, p300 acetylates and increases the activity of p53 which leads to increased apoptotic activity(3). Cardiotoxicity results because the heart has limited mechanisms to dispose of ROSs(4), and because cardiomyocytes have a low turnover rate(5). Purpose We examined the p300 inhibitor Theracurmin (THR)(6,7) as a candidate to prevent DOX cardiotoxicity using an in-vivo mouse model, and sought to elucidate the underlying molecular mechanisms using in-vitro studies. Methods C57BL/6 mice were pre-treated with THR or vehicle (as control), then administered DOX or vehicle. Cardiac function was evaluated by echocardiography and cardiac catheterization. In vitro studies used cardiac fibroblasts (FBs) and cardiac endothelial cells (ECs) pre-treated with THR, then with DOX, as in the animal studies. Western blotting and rt-qPCR was used to quantify protein and mRNA levels of genes in the p53-p300 pathway or related to apoptosis, oxidative stress, and cell cycle control. Results DOX-treated mice showed significant decreases in left ventricular (LV) ejection fraction (LVEF, p=0.0004), fractional shortening (FS, p=0.0012), and a significant increase in end-systolic volume (ESV, p=0.0004). We also showed a significant decrease in anterior (LVAW;d, p=0.005) and posterior (LVPW;d, p=0.005) wall thickness of the LV in these mice. However, when mice were pre-treated with THR prior to DOX administration, we saw significant increases in LVEF (p=0.045) and LVPW;d (p=0.015), and a significant decrease in ESV (p=0.0047) compared to those that were not pre-treated. In-vitro analyses demonstrated a significant increase in cleaved caspase 3 protein expression in DOX-treated ECs (p=0.007), that was not prevented by THR pre-treatment. In DOX-treated FBs, we saw a reduction in AKT mRNA (p=0.017) and increases in BAX 9p=0.008), P21 (p=0.0001), and GADD45 (0.037) mRNA expression. In DOX-treated ECs, we also saw significant increases in BAX (p=0.015) and P21 (p=0.011) mRNA expression. These changes in mRNA expression were not reversed with THR pre-treatment in either cell type. Conclusion THR pre-treatment successfully mitigated functional deficits and structural deficits associated with DOX cardiotoxicity. In-vitro studies show that THR pre-treatment was not sufficient to significantly prevent changes in key molecular targets associated with apoptosis. Further studies are required to elucidate the mechanism behind the observed functional changes.Abstract OverviewAbstract Results

Effects of BET Inhibition on lung micro-environmental changes in obesity-related pulmonary Arterial Hypertension (PAH)

Abstract Background Obesity is emerging as a pivotal risk factor for pulmonary arterial hypertension (PAH) but the underlying molecular mechanisms are poorly explored. The lung microenvironment, namely the cell-cell communication among fibroblasts, immune and endothelial cells, may significantly impact on vascular structure and function thus promoting microvascular disease in this setting. Obesity-induced chromatin modifications lead to maladaptive transcriptional programs altering cell function in different organs. Apabetalone (APA), a selective inhibitor of bromodomain and extra-terminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating gene expression. Purpose To determine whether a new epi-drug, namely APA, affects the lung microenvironment in obesity-related PAH. Methods Mice were fed a normal diet (ND, control) or high-fat-diet (HFD) and L-NAME for 15 weeks to induce obesity-related PAH (obPAH). Echocardiography was performed. Pathways regulating obPAH were identified by single-cell nuclei RNA sequencing in lung specimens. Primary endothelial cells (ECs) and Fibroblasts (mFs) were freshly isolated from obPAH lungs and treated with APA for 48 hours. Cells and lung samples were used for molecular biology and histology. Cellular migration was investigated by in vitro gap closure. Results Obese mice displayed PAH as shown by increased pulmonary artery (PA) pressure, PA resistance, right ventricular wall thickness and reduced PA acceleration time. Of note, chronic APA treatment prevented PAH-related features in obese mice. To determine the cell types affected by APA, we performed scRNAseq. We found that fibroblast and endothelial cells had the highest enrichment of genes whose expression significantly correlated with echo-determined PAH features. ECs and mFs from obese mice displayed an altered phenotype whereas treatment with APA rescued obesity-driven ECs and mFs dysfunction. In obPAH ECs, we found increased expression of NF-kB p65-related inflammatory genes (IL-1β, IL-6, TNF-alpha) as well as enhanced p53 signaling. Moreover, obPAH-ECs expressed high levels of hypoxia-inducible factor (HIF1-α) and NADPH-oxidase-NOX4 with subsequent increase in ROS generation. Furthermore, APA treatment protected against cellular inflammation, senescence and oxidative stress. To evaluate the effect of APA and the paracrine response of fibroblasts, we performed a scratch assay on control mFs exposed to TGF-β or conditioned media collected from obPAH-ECs with and without APA. In both experimental settings, APA affected gap closure, suggesting that the treatment influences fibroblast migration through both a direct effect and paracrine mechanisms. Conclusions APA is able to reset the lung micro-envirnment in obesity thus reducing inflammation and senescence. BET inhibitors could represent potential therapeutic approaches in this setting.

Genistein inhibited endocytosis and fibrogenesis in keloid via CTGF signaling pathways

BackgroundThis study aimed to evaluate soy isoflavones' effect and potential use—specifically genistein—in treating human keloid fibroblast cells (KFs) and in a keloid tissue culture model.MethodsTo investigate the effects of genistein on keloid, a wound-healing assay was performed to detect cell migration. Flow cytometry was used to measure apoptosis. Western blotting and immunofluorescence staining were performed to detect the expression of target proteins. KF tissues were isolated, cultured, and divided into the control, silenced connective tissue growth factor (CTGF) proteins, and shNC (negative control) groups.ResultsGenistein suppressed cell proliferation and migration, triggering the cell cycle at the G2/M phase and increasing the expression of p53 dose-dependent in keloids. Genistein inhibited the expression of COL1A1, FN, and CTGF mRNA and protein. Knockdown CTGF reduced the migrated ability in KFs. Genistein also abated TGF-β1-induced keloid fibrosis through the endocytosis model. Separated and cultured the keloid patient’s tissues decreased the cell migration ability by genistein treatment and was time-dose dependent.ConclusionsThis study indicated that genistein-induced p53 undergoes cell cycle arrest via the CTGF pathway-inhibited keloid cultured cells, and genistein suppressed the primary keloid cell migration, suggesting that our research provides a new strategy for developing drugs for treating keloids.

Fabrication and Characterization of Biocompatible Multilayered Elastomer Hybrid with Enhanced Water Permeation Resistance for Packaging of Implantable Biomedical Devices

This study presents the synthesis and characterization of hexadecyl-modified SiO2 (HD-SiO2) nanoparticles and their application in the fabrication of a multilayered elastomer hybrid sheet to enhance water resistance in implantable biomedical devices. The surface modification of SiO2 nanoparticles was confirmed via FT-IR and TGA analyses, showing the successful grafting of hydrophobic hexadecyl groups. FE-SEM and DLS analyses revealed spherical HD-SiO2 nanoparticles with an average size of 360 nm. A multilayered elastomer hybrid sheet, consisting of a PDMS-based circuit-protecting body, a water resistance layer of HD-SiO2, a planarization layer, and a biocompatible layer of polydopamine, was fabricated and characterized. The water resistance layer exhibited superhydrophobic properties, with a water contact angle of 154.7° and a 27% reduction in water vapor transmission rate (WVTR) compared to the circuit-protecting body alone. The device packaged with both the circuit-protecting body and water resistance layer demonstrated a tenfold increase in operational lifespan in water medium compared to the device without the water resistance layer. Cytotoxicity and cell proliferation tests on human dermal fibroblast cells (HDFn) confirmed the biocompatibility of the multilayered sheet, with no significant cytotoxicity observed over 48 h.