Development and validation of novel keloid-derived immortalized fibroblast cell lines.

Keloids are a common connective tissue disorder with an ill-understood etiopathogenesis and no effective treatment. This is exacerbated because of the absence of an animal model. Patient-derived primary keloid cells are insufficient as they age through passaging and have a limited supply. Therefore, there is an unmet need for development of a cellular model that can consistently and faithfully represent keloid's pathognomic features. In view of this, we developed keloid-derived immortalized fibroblast (KDIF) cell lines from primary keloid fibroblasts (PKF) by transfecting the human telomerase reverse transcriptase (hTERT) gene. The TERT gene encodes the catalytic subunit of the telomerase enzyme, which is responsible for maintaining the cellular replicative potential (cellular immortalization). Primary fibroblasts from keloid-specific lesional (peripheral, middle, and top) as well as extralesional sites were isolated and evaluated for cell line development and comparative cellular characteristics by employing qRT-PCR and immunofluorescence staining. Moreover, the immortalized behavior of KDIF cell lines was evaluated by comparing with cutaneous fibrosarcoma and dermatofibrosarcoma protuberans cell lines. Stable KDIF cell lines with elevated expression of hTERT exhibited the cellular characteristics of site-specific keloid fibroblasts. Histochemical staining for ß-galactosidase revealed a significantly lower number of ß-gal-positive cells in all three KDIF cell lines compared with that in PKFs. The cell growth curve pattern was studied over 10 passages for all three KDIF cell lines and was compared with the control groups. The results showed that all three KDIF cell lines grew significantly faster and obtained a fast growing characteristic as compared to primary keloid and normal fibroblasts. Phenotypic behavior in growth potential is an indication of hTERT-mediated immortalized transformation. Cell migration analysis revealed that the top and middle KDIF cell lines exhibited similar migration trend as site-specific PKFs. Notably, peripheral KDIF cell line showed significantly enhanced cell migration in comparison to the primary peripheral fibroblasts. All KDIF cell lines expressed Collagen I protein as a keloid-associated fibrotic marker. Functional testing with triamcinolone inhibited cell migration in KDIF. ATCC short tandem repeat profiling validated the KDIF as keloid representative cell line. In summary, we provide the first novel KDIF cell lines. These cell lines overcome the limitations related to primary cell passaging and tissue supply due to immortalized features and present an accessible and consistent experimental model for keloid research.

DCM-Spheroid Morphs Express PADs and Citrullinated Cytoskeletal Proteins.

During investigating the role of peptidylarginine deiminase (PAD) enzymes in dilated cardiomyopathy (DCM), we observed unique spheroid formation in DCM-myofibroblasts that distinguished them from normal cardiac myofibroblasts. The present study aimed to assess the presence of PADs, the extracellular matrix (ECM), and citrullination in DCM spheroids using immunofluorescence staining and imaging techniques. The results revealed that spheroids derived from DCM-myofibroblasts displayed a more distinctive, tightly packed structure compared with those derived from human cardiac fibroblasts. DCM spheroids showed abundant protein expression of the PAD 2, 3, and 4 enzymes. Notably, increased Ki67 protein expression was associated with increased proliferation in DCM spheroids. Cytoskeletal proteins such as Col-1A, vimentin, α-SMA, and F-actin were highly abundant in DCM spheroids. Furthermore, DCM spheroids contained citrullinated cytoskeletal proteins, mainly citrullinated vimentin and citrullinated fibronectin. These observations supported the occurrence of PAD-mediated citrullination of ECM proteins in DCM spheroids. Collectively, these findings describe the distinctive features of DCM spheroids, representing the cellular characteristics of DCM myofibroblasts. Therefore, DCM spheroids can serve as an in vitro model for further investigations of disease morphology and therapeutic efficacy.

Treatment of human cardiac fibroblasts with the protein arginine deiminase inhibitor BB-Cl-amidine activates the Nrf2/HO-1 signaling pathway.

BACKGROUND: Cardiac fibrosis contributes to end-stage extracellular matrix remodeling and heart failure (HF). Cardiac fibroblasts (CFs) differentiate into myofibroblasts (myoFbs) to preserve the structural integrity of the heart; however, the molecular mechanisms regulating CF transdifferentiation remain poorly understood. Protein arginine deiminase (PAD), which converts arginine to citrulline, has been shown to play a role in myocardial infarction, fibrosis, and HF. This study aimed to investigate the role of PAD in CF differentiation to myoFbs and identify the citrullinated proteins that were associated with phenotypic changes in CFs. RESULTS: Gene expression analysis showed that PAD1 and PAD2 isoforms, but not PAD4 isoforms, were abundant in both CFs and myoFbs, and PAD1 was significantly upregulated in myoFbs. The pan-PAD inhibitor BB-Cl-amidine (BB-Cl) downregulated the mRNA expression of PAD1 and PAD2 as well as the protein expression of the fibrosis marker COL1A1 in CFs and myoFbs. Interestingly, a proteomic approach pointed to the activation of the Nrf2/HO-1 signaling pathway upon BB-Cl treatment in CFs and myoFbs. BB-Cl administration resulted in the upregulation of HO-1 at both the gene and protein levels in CFs and myoFbs. Importantly, the protein citrullination landscape of CFs consisting of 86 novel citrullination sites associated with focal adhesion (FN1(R1054)), inflammation (TAGLN(R12)) and DNA replication (EEF2(R767)) pathways was identified. CONCLUSIONS: In summary, we revealed that BB-Cl treatment resulted in increased HO-1 expression via the Nrf2 pathway, which could prevent excessive tissue damage, thereby leading to substantial clinical benefits for the treatment of cardiac fibrosis.

The Role of Serotonin during Skin Healing in Post-Thermal Injury.

Post-burn trauma significantly raises tissue serotonin concentration at the initial stages of injury, which leads us to investigate its possible role in post burn wound healing. Therefore, we planned this study to examine the role of serotonin in wound healing through in vitro and in vivo models of burn injuries. Results from in vitro analysis revealed that serotonin decreased apoptosis and increased cell survival significantly in human fibroblasts and neonatal keratinocytes. Cellular proliferation also increased significantly in both cell types. Moreover, serotonin stimulation significantly accelerated the cell migration, resulting in narrowing of the scratch zone in human neonatal keratinocytes and fibroblasts cultures. Whereas, fluoxetine (a selective serotonin reuptake inhibitor) and ketanserin (serotonin receptor 2A inhibitor) reversed these effects. Scald burn mice model (20% total body surface area) showed that endogenous serotonin improved wound healing process in control group, whereas fluoxetine and ketanserin treatments (disruptors of endogenous serotonin stimulation), resulted in poor reepithelization, bigger wound size and high alpha smooth muscle actin (α-SMA) count. All of these signs refer a prolonged differentiation state, which ultimately exhibits poor wound healing outcomes. Collectively, data showed that the endogenous serotonin pathway contributes to regulating the skin wound healing process. Hence, the results of this study signify the importance of serotonin as a potential therapeutic candidate for enhancing skin healing in burn patients.

5-HT1A Receptor Function Makes Wound Healing a Happier Process.

Skin wound healing is a multistage phenomenon that is regulated by cell-cell interplay and various factors. Endogenous serotonin is an important neurotransmitter and cytokine. Its interaction with the serotonin 1A receptor (5-HTR1A) delivers downstream cellular effects. The role of serotonin (5-hydroxytryptamine, 5-HT) and the 5-HT1A receptor has been established in the regeneration of tissues such as the liver and spinal motor neurons, prompting the investigation of the role of 5-HT1A receptor in skin healing. This study assessed the role of 5-HT1A receptor in excisional wound healing by employing an excisional punch biopsy model on 5-Ht1a receptor knockout mice. Post-harvest analysis revealed 5-Ht1a receptor knockout mice showed impaired skin healing, accompanied by a greater number of F4/80 macrophages, which prolongs the inflammatory phase of wound healing. To further unravel this phenomenon, we employed the 5-HT1A receptor agonist [(R)-(+)-8-Hydroxy-DPAT hydrobromide] as a topical cream treatment in an excisional punch biopsy model. The 5-HT1A receptor agonist treated group showed a smaller wound area, scar size, and improved neovascularization, which contributed to improve healing outcomes as compared to the control. Collectively, these findings revealed that serotonin and 5-HT1A receptor play an important role during the healing process. These findings may open new lines of investigation for the potential treatment alternatives to improve skin healing with minimal scarring.

A comparative ethno-botanical study of Cholistan (an arid area) and Pothwar (a semi-arid area) of Pakistan for traditional medicines.

BACKGROUND: The present study is intended to compare and document the therapeutic flora, their remedial use, and the traditional knowledge used frequently by the residents of the Cholistan desert and Pothwar (Potohar) Plateau of Punjab, Pakistan. The old endemic remedies of these areas are diminishing due to lack of qualitative and quantitative research. METHODS: The data was generated by unstructured-interviews, informal meetings, open-ended conversations and group discussions with local people and traditional health healers of the study area. Reported literature was also utilized. RESULTS: The study recorded a list of various medicinal plants used as traditional medicines by local people. Total 86 numbers of plant species belonging to 38 families and 67 plant species belonging to 29 families have been reported in the Pothwar and Cholistan respectively. Only 10.5% of similar plant species were present in the studied areas. CONCLUSION: The investigation revealed that the local people of study areas inherit a rich traditional knowledge but there is great danger of losing this wealth of knowledge in the near future. Documentation of the knowledge exclusively from desert area of Cholistan, Pakistan is unique information in its nature. The study presents the undocumented knowledge worth recognition that will not only help in conservation of medicinal plant species but will highlight the pharmacological capacity for improved human healthcare regarding many common ailments.