Doxorubicin-Induced Modulation of TGF-β Signaling Cascade in Mouse Fibroblasts: Insights into Cardiotoxicity Mechanisms

Doxorubicin (DOX)-induced cardiotoxicity has been widely observed, yet the specific impact on cardiac fibroblasts is not fully understood. Additionally, the modulation of the transforming growth factor beta (TGF-β) signaling pathway by DOX remains to be fully elucidated. This study investigated DOX’s ability to modulate the expression of genes and proteins involved in the TGF-β signaling cascade in mouse fibroblasts from two sources by assessing the impact of DOX treatment on TGF-β inducible expression of pivotal genes and proteins within fibroblasts. Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX in the presence of TGF-β1 to assess changes in protein levels by western blot and changes in mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Our results revealed a dose-dependent reduction in cellular communication network factor 2 (CCN2) protein levels upon DOX treatment in both NIH3T3 and CFs, suggesting an antifibrotic activity by DOX in these fibroblasts. However, DOX only inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells but not in CFs. In addition, we observed that DOX treatment reduced the expression of BMP1 in NIH3T3 but not primary cardiac fibroblasts. No significant changes in SMAD2 protein expression and phosphorylation in either cells were observed after DOX treatment. Finally, DOX inhibited the expression of Atf4 gene and increased the expression of Cdkn1a, Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1 genes in NIH3T3 cells but not CFs, indicating the potential for cell-specific responses to DOX and its modulation of the TGF-β signaling pathway

Advanced Atomic Force Microscopy for BioMaterials Research

Optical microscopy uses the interactions between light and materials to provide images of the microscopic world. It is widely employed in science to study the behavior and properties of microscopic organisms and cells. Atomic force microscopy (AFM) is a technique for obtaining images of the surfaces of materials at the atomic to micrometer scales. AFM operates by rastering an ultra-sharp needle across a sample surface and recording the height of the needle at each position. While AFM can provide atomic resolution images of the contours (topography) of a surface, it can also perform extremely sensitive measurements of surface mechanical properties. By fabricating custom AFM probes, the mechanical properties of specific locations of living cells can be studied and manipulated. In addition, high-speed imaging of biological materials can provide images of changes to cellular surfaces in response to chemical or electrical signals. This poster will present examples and applications of advanced AFM capabilities for research in biomaterials available in the Boise State University Surface Science Laboratory

Effects of Doxorubicin on Extracellular Matrix Regulation in Primary Cardiac Fibroblasts from Mice

Objective Doxorubicin (DOX) is a highly effective chemotherapeutic used to treat many adult and pediatric cancers. However, its use is limited due to a dose-dependent cardiotoxicity, which can lead to lethal cardiomyopathy. In contrast to the extensive research efforts on toxic effects of DOX in cardiomyocytes, its effects and mechanisms on cardiac extracellular matrix (ECM) homeostasis and remodeling are poorly understood. In this study, we examined the potential effects of DOX on cardiac ECM to further our mechanistic understanding of DOX-induced cardiotoxicity. Results DOX-induced significant down-regulation of several ECM related genes in primary cardiac fibroblasts, including Adamts1, Adamts5, Col4a1, Col4a2, Col5a1, Fbln1, Lama2, Mmp11, Mmp14, Postn, and TGFβ. Quantitative proteomics analysis revealed significant global changes in the fibroblast proteome following DOX treatment. A pathway analysis using iPathwayGuide of the differentially expressed proteins revealed changes in a list of biological pathways that involve cell adhesion, cytotoxicity, and inflammation. An apparent increase in Picrosirius red staining indicated that DOX-induced an increase in collagen production in cardiac primary fibroblasts after 3-day treatment. No significant changes in collagen organization nor glycoprotein production were observed

Doxorubicin Modulates Autophagy in a Cell-Type Dependent Manner: A Potential Mechanism for Doxorubicin Induced Cardiotoxicity

Doxorubicin is a highly effective chemotherapeutic, but its use is limited due to dose-dependent cardiotoxicity, which can result in lethal cardiomyopathy. Although oxidative stress leading to apoptosis (cell death) is generally accepted as the principal mechanism for doxorubicin-induced cardiotoxicity, antioxidant interventions have largely failed in pre-clinical and clinical trials. Other novel mechanisms that could explain this effect include autophagy. Autophagy is a highly conserved process of self-degradation of cellular components in response to cellular stress and signals such as starvation, growth factor deprivation, and pathogen infection. Interference with this process may also lead to the induction of apoptosis and downstream cardiotoxic symptoms. Previous studies have demonstrated doxorubicin influences autophagy in cardiomyocytes. However, little is known about the effects of this chemotherapeutic in fibroblasts. Fibroblasts are the largest cell population in the heart and the main cell type responsible for the synthesis, deposition, and degradation of cardiac extracellular matrix (ECM). Through use of cell culture, western blotting, and reverse transcription quantitative polymerization chain reaction (RT-qPCR) techniques, we studied the effects of doxorubicin on autophagy by the monitored expression of known autophagy markers. Expression of autophagy markers P62 and LC3B analyzed by western blotting suggests doxorubicin induces autophagy in NIH 3T3 fibroblast cells. A similar trend was not obvious in mouse primary cardiac fibroblasts, signifying this effect may be cell-type dependent. Preliminary RT-qPCR analysis also indicates that doxorubicin modulates the expression of several genes in autophagy pathway. With further study, autophagy could be a potential target for mitigating doxorubicin-induced cardiotoxicity

Effects of Simulated Microgravity on Mesenchymal Stem Cell Mechanosensitivity

Essentially all cells are sensitive to mechanical signals, providing the tissue and organismal level of adaptability to mechanical challenges. At cell level this adaptability is reflected as increased cytoskeletal connectivity. Our findings indicate that, while simulated microgravity (sMG) decreases cellular connectivity, application of known exercise mimetic, low intensity vibration (LIV) improves it. Here we will test the hypothesis that application of LIV during sMG will improve MSC responsiveness to high magnitude strain (HMS). To allow application of sMG on stretchable silicone membranes, cells will be encapsulated in Collagen Type I. HMS will be applied using a Flexcell-5000 device at 2%, 0.2Hz, MSCs will be subjected to sMG with or without LIV for 3 days. Following the sMG±LIV protocols, cells wills be subjected to HMS for 20 minutes. The intensity of the HMS response will be measured by the acute FAK (tyr 397) and Akt (Ser 473) phosphorylation events

Doxorubicin-induced modulation of TGF-β signaling cascade in mouse fibroblasts: insights into cardiotoxicity mechanisms

Abstract Doxorubicin (DOX)-induced cardiotoxicity has been widely observed, yet the specific impact on cardiac fibroblasts is not fully understood. Additionally, the modulation of the transforming growth factor beta (TGF-β) signaling pathway by DOX remains to be fully elucidated. This study investigated DOX’s ability to modulate the expression of genes and proteins involved in the TGF-β signaling cascade in mouse fibroblasts from two sources by assessing the impact of DOX treatment on TGF-β inducible expression of pivotal genes and proteins within fibroblasts. Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX in the presence of TGF-β1 to assess changes in protein levels by western blot and changes in mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Our results revealed a dose-dependent reduction in cellular communication network factor 2 (CCN2) protein levels upon DOX treatment in both NIH3T3 and CFs, suggesting an antifibrotic activity by DOX in these fibroblasts. However, DOX only inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells but not in CFs. In addition, we observed that DOX treatment reduced the expression of BMP1 in NIH3T3 but not primary cardiac fibroblasts. No significant changes in SMAD2 protein expression and phosphorylation in either cells were observed after DOX treatment. Finally, DOX inhibited the expression of Atf4 gene and increased the expression of Cdkn1a, Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1 genes in NIH3T3 cells but not CFs, indicating the potential for cell-specific responses to DOX and its modulation of the TGF-β signaling pathway

Investigating the Cardiotoxic Behavior of Doxorubicin on the ECM Through CCN2 Expression in Cardiac Fibroblasts

Cellular Communication Network Factor 2 (CCN2) is a matricellular protein encompassing many cellular functions, including extracellular matrix (ECM) deposition. The role of CCN2 has been investigated in numerous studies, but one region of CCN2 that has not been fully investigated is the role of CCN2 when exposed to cardiotoxic chemotherapeutics and how it affects the cardiac ECM. During chronic cardiac inflammation, CCN2 is activated, which eventually leads to cardiac fibrosis and remodeling. Consequently, this result has been shown to markedly reduce the efficacy of cardiac function, contributing to cardiomyopathy when severe. Doxorubicin (DOX) is an anthracycline chemotherapeutic commonly used to treat many cancers, such as breast and blood cancers. Fibroblasts are one of the leading cell lines to partake in ECM regulation, especially in the heart. In this study, we investigated the effects of DOX on CCN2 expression in NIH3T3 mouse embryonic fibroblast and primary mouse cardiac fibroblast. We administered DOX treatments and performed protein and RNA isolation on our cell lines for the use of western blot and RT-qPCR, respectively. A focus was placed on CCN2 as well as other proteins established to promote CCN2 protein and gene expression. Lastly, we also investigated ECM proteins commonly expressed in the presence of CCN2. As a result, our study explores DOX’s adverse effects on the cardiac extracellular matrix by means of disruption of ECM depositor CCN2

Effects of Doxorubicin on Cardiac Fibroblasts and the Extracellular Matrix

Cardiotoxicity has been associated with various types of chemotherapeutic drugs contributing to a plethora of cardiac insults and is a significant side effect when treating cancer. Many highly effective anticancer drugs are severely dose dependent, and at higher doses can lead to: cardiac arrhythmias, hypertension, and lethal cardiomyopathy. A well known example of this cardiotoxic side effect is Doxorubicin, a common chemotherapeutic used to treat cancers of the breast, ovary, bladder, and thyroid. Extensive research has shown that high doses of doxorubicin detrimentally alters the normal function of cardiac fibroblasts and cardiomyocytes. In contrast to the extensive research on the toxic effects of chemotherapeutics like doxorubicin in cardiomyocytes, little is known on the effects in cardiac fibroblasts and mechanisms of these drugs on the cardiac extracellular matrix (cECM). We show that doxorubicin has a direct impact on cardiac fibroblasts and in turn the function of the cECM, indicating that the cECM plays an important role in cardiac toxicity induced by doxorubicin

Valinomycin–mediated Cation Transport in Planar Bilayer Lipid Membranes

Valinomycin is a natural dodecadepsipetide ionophore largely used for selective K+ transport across artificial and natural lipid membranes, and as an antibiotic. As a transporter, valinomycin passively moves K+ ions across membrane down the electrochemical gradient. It is well known that valinomycin selectivity for K+ is ~ 100,000 greater then the selectivity measured for Na+, which makes the carrier an excellent tool for exploring transport phenomena in living cells, where the ions are major cations. However, little is known about valinomycin’s capability to transport other monovalent cations, or how the lipid composition of the target membrane affect membrane binding and transport properties. Therefore, our studies focused on understanding the valinomycin’s transport properties in artificial membrane systems comprising planar lipid membranes and electrophysiology measurements