Proteome analysis, bioinformatic prediction and experimental evidence revealed immune response down-regulation function for serum-starved human fibroblasts.

Emerging evidence indicates that fibroblasts play pivotal roles in immunoregulation by producing various proteins under health and disease states. In the present study, for the first time, we compared the proteomes of serum-starved human skin fibroblasts and peripheral blood mononuclear cells (PBMCs) using Nano-LC-ESI-tandem mass spectrometry. This analysis contributes to a better understanding of the underlying molecular mechanisms of chronic inflammation and cancer, which are intrinsically accompanied by growth factor deficiency.The proteomes of starved fibroblasts and PBMCs consisted of 307 and 294 proteins, respectively, which are involved in lymphocyte migration, complement activation, inflammation, acute phase response, and immune regulation. Starved fibroblasts predominantly produced extracellular matrix-related proteins such as collagen/collagenase, while PBMCs produced focal adhesion-related proteins like beta-parvin and vinculin which are involved in lymphocyte migration. PBMCs produced a more diverse set of inflammatory molecules like heat shock proteins, while fibroblasts produced human leukocytes antigen-G and -E that are known as main immunomodulatory molecules. Fifty-four proteins were commonly found in both proteomes, including serum albumin, amyloid-beta, heat shock cognate 71 kDa, and complement C3. GeneMANIA bioinformatic tool predicted 418 functions for PBMCs, including reactive oxygen species metabolic processes and 241 functions for starved fibroblasts such as antigen processing and presentation including non-classical MHC -Ib pathway, and negative regulation of the immune response. Protein-protein interactions network analysis indicated the immunosuppressive function for starved fibroblasts-derived human leucocytes antigen-G and -E. Moreover, in an in vitro model of allogeneic transplantation, the immunosuppressive activity of starved fibroblasts was experimentally documented. Conclusion: Under serum starvation-induced metabolic stress, both PBMCs and fibroblasts produced molecules like heat shock proteins and amyloid-beta, which can have pathogenic roles in auto-inflammatory diseases such as rheumatoid arthritis, type 1 diabetes mellitus, systemic lupus erythematosus, aging, and cancer. However, starved fibroblasts showed immunosuppressive activity in an in vitro model of allogeneic transplantation, suggesting their potential to modify such adverse reactions by down-regulating the immune system.

Ninety-six-hour starved peripheral blood mononuclear cell supernatant inhibited LA7 breast cancer stem cells induced tumor via reduction in angiogenesis and alternations in Gch1 and Spr expressions.

Introduction: The microenvironment of solid tumors such as breast cancer is heterogeneous and complex, containing different types of cell, namely, cancer stem cells and immune cells. We previously reported the immunoregulatory behavior of the human immune cell in a solid tumor microenvironment-like culture under serum starvation stress for 96 h. Here, we examined the effect of this culture-derived solution on breast cancer development in rats. Method: Ninety-six-hour starved PBMCs supernatant (96 h-SPS) was collected after culturing human PBMCs for 96 h under serum starvation condition. Breast cancer stem cells, LA7 cell line, was used for in vitro study by analyzing gene expression status and performing cytotoxicity, proliferation, scratch wound healing assays, followed by in vivo tumor induction in three groups of mature female Sprague Dawley rats. Animals were treated with 96 h-SPS or RPMI and normal saline as control, n = 6 for each group. After biochemical analysis of iron, lactate, and pH levels in the dissected tumors, Ki67 antigen expression, angiogenesis, and necrosis evaluation were carried out. Metabolic-related gene expression was assessed using RT-qPCR. Moreover, 96 h-SPS composition was discovered by Nano-LC-ESI-MS/MS. Results: 96 h-SPS solution reduced the LA7 cell viability, proliferation, and migration and Gch1 and Spr genes expression in vitro (p< 0.05), whereas stemness gene Oct4 was upregulated (p< 0.01). The intracellular lactate was significantly decreased in the 96 h-SPS treated group (p = 0.007). In this group, Gch1 and Spr were significantly downregulated (p< 0.05), whereas the Sox2 and Oct4 expression was not changed significantly. The number of vessels and mitosis (Ki67+ cells) in the 96 h-SPS-treated group was significantly reduced (p = 0.024). The increased rate of necrosis in this group was statistically significant (p = 0.04). Last, proteomics analysis revealed candidate effectors' components of 96 h-SPS solution. Conclusion: 96 h-SPS solution may help to prevent cancer stem cell mediated tumor development. This phenomenon could be mediated through direct cytotoxic effects, inhibition of cell proliferation and migration in association with reduction in Gch1 and Spr genes expression, angiogenesis and mitosis rate, and necrosis augmentation. The preliminary data obtained from the present study need to be investigated on a larger scale and can be used as a pilot for further studies on the biology of cancer development.

Patients with Coronary Artery Disease Have Lower Levels of Antibody to Heat-Stressed Fibroblast Derived Proteins, versus Normal Subjects.

Cellular stress response plays an important role in the pathophysiology of coronary artery disease (CAD). Inhibition of cellular stress may provide a novel clinical approach regarding the diagnosis and treatment of CAD. Fibroblasts constitute 60-70% of cardiac cells and have a crucial role in cardiovascular function. Hence, the aim of this study was to show a potential therapeutic application of proteins derived from heat-stressed fibroblast in CAD patients. Fibroblasts were isolated from the foreskin and cultured under heat stress conditions. Surprisingly, 1.06% of the cells exhibited a necrotic death pattern. Furthermore, heat-stressed fibroblasts produced higher level of total proteins than control cells. In SDS-PAGE analysis, a 70 kDa protein band was observed in stressed cell culture supernatants which appeared as two acidic spots with close pI in the two-dimensional electrophoresis. To evaluate the immunogenic properties of fibroblast-derived heat shock proteins (HSPs), the serum immunoglobulin-G (IgG) was measured by ELISA in 50 CAD patients and 50 normal subjects who had been diagnosed through angiography. Interestingly, the level of anti-HSP antibody was significantly higher in non-CAD individuals in comparison with the patient's group (p < 0.05). The odds ratio for CAD was 5.06 (95%CI = 2.15-11.91) in cut-off value of 30 AU/mL of anti-HSP antibody. Moreover, ROC analysis showed that anti-HSP antibodies had a specificity of 74% and a sensitivity of 64%, which is almost equal to 66% sensitivity of exercise stress test (EST) as a CAD diagnostic method. These data revealed that fibroblast-derived HSPs are suitable for the diagnosis and management of CAD through antibody production.