Mechanical stretch leads to increased caveolin-1 content and mineralization potential in extracellular vesicles from vascular smooth muscle cells.

BACKGROUND: Hypertension-induced mechanical stress on vascular smooth muscle cells (VSMCs) is a known risk factor for vascular remodeling, including vascular calcification. Caveolin-1 (Cav-1), an integral structural component of plasma membrane invaginations, is a mechanosensitive protein that is required for the formation of calcifying extracellular vesicles (EVs). However, the role of mechanics in Cav-1-induced EV formation from VSMCs has not been reported. RESULTS: Exposure of VSMCs to 10% mechanical stretch (0.5 Hz) for 72 h resulted in Cav-1 translocation into non-caveolar regions of the plasma membrane and subsequent redistribution of Cav-1 from the VSMCs into EVs. Inhibition of Rho-A kinase (ROCK) in mechanically-stimulated VSMCs exacerbated the liberation of Cav-1 positive EVs from the cells, suggesting a potential involvement of actin stress fibers in this process. The mineralization potential of EVs was measured by incubating the EVs in a high phosphate solution and measuring light scattered by the minerals at 340 nm. EVs released from stretched VSMCs showed higher mineralization potential than the EVs released from non-stretched VSMCs. Culturing VSMCs in pro-calcific media and exposure to mechanical stretch increased tissue non-specific alkaline phosphatase (ALP), an important enzyme in vascular calcification, activity in EVs released from the cells, with cyclic stretch further elevating EV ALP activity compared to non-stretched cells. CONCLUSION: Our data demonstrate that mechanical stretch alters Cav-1 trafficking and EV release, and the released EVs have elevated mineralization potential.

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models.

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.

Membrane Remodeling of Human-Engineered Cardiac Tissue by Chronic Electric Stimulation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show immature features, but these are improved by integration into 3D cardiac constructs. In addition, it has been demonstrated that physical manipulations such as electrical stimulation (ES) are highly effective in improving the maturation of human-engineered cardiac tissue (hECT) derived from hiPSC-CMs. Here, we continuously applied an ES in capacitive coupling configuration, which is below the pacing threshold, to millimeter-sized hECTs for 1-2 weeks. Meanwhile, the structural and functional developments of the hECTs were monitored and measured using an array of assays. Of particular note, a nanoscale imaging technique, scanning ion conductance microscopy (SICM), has been used to directly image membrane remodeling of CMs at different locations on the tissue surface. Periodic crest/valley patterns with a distance close to the sarcomere length appeared on the membrane of CMs near the edge of the tissue after ES, suggesting the enhanced transverse tubulation network. The SICM observation is also supported by the fluorescence images of the transverse tubulation network and α-actinin. Correspondingly, essential cardiac functions such as calcium handling and contraction force generation were improved. Our study provides evidence that chronic subthreshold ES can still improve the structural and functional developments of hECTs.

Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair.

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

Scanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA Damage.

Oxidative stress can damage organs, tissues, and cells through reactive oxygen species (ROS) by oxidizing DNA, proteins, and lipids, thereby resulting in diseases. However, the underlying molecular mechanisms remain to be elucidated. In this study, employing scanning ion conductance microscopy (SICM), we explored the early responses of human embryonic kidney (HEK293H) cells to oxidative DNA damage induced by potassium chromate (K2CrO4). We found that the short term (1-2 h) exposure to a low concentration (10 µM) of K2CrO4 damaged the lipid membrane of HEK293H cells, resulting in structural defects and depolarization of the cell membrane and reducing cellular secretion activity shortly after the treatment. We further demonstrated that the K2CrO4 treatment decreased the expression of the cytoskeleton protein, ß-actin, by inducing oxidative DNA damage in the exon 4 of the ß-actin gene. These results suggest that K2CrO4 caused oxidative DNA damage in cytoskeleton genes such as ß-actin and reduced their expression, thereby disrupting the organization of the cytoskeleton beneath the cell membrane and inducing cell membrane damages. Our study provides direct evidence that oxidative DNA damage disrupted human cell membrane integrity by deregulating cytoskeleton gene expression.

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase ß and a de novo DNA Methyltransferase.

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from -189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase ß (pol ß). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol ß and the de novo DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and de novo DNA methylation.