BACKGROUND: VWF (von Willebrand factor) is an endothelial-specific procoagulant protein with a major role in thrombosis. Aging is associated with increased circulating levels of VWF, which presents a risk factor for thrombus formation. METHODS: Circulating plasma, cellular protein, and mRNA levels of VWF were determined and compared in young and aged mice. Major organs were subjected to immunofluorescence analyses to determine the vascular pattern of VWF expression and the presence of platelet aggregates. An in vitro model of aging, using extended culture time of endothelial cells, was used to explore the mechanism of age-associated increased VWF levels. RESULTS: Increased circulating plasma levels of VWF with elevated levels of larger multimers, indicative of VWF functional activity, were observed in aged mice. VWF mRNA and cellular protein levels were significantly increased in the brains, lungs, and livers but not in the kidneys and hearts of aged mice. Higher proportion of small vessels in brains, lungs, and livers of aged mice exhibited VWF expression compared with young, and this was concomitant with increased platelet aggregate formation. Prolonged culture of endothelial cells resulted in increased cell senescence that correlated with increased VWF expression; VWF expression was specifically detected in senescent cultured endothelial cells and abolished in response to p53 knockdown. A significantly higher proportion of VWF expressing endothelial cells in vivo exhibited senescence markers SA-beta-Gal (senescence-associated beta-galactosidase) and p53 in aged mouse brains compared with that of the young. CONCLUSIONS: Aging elicits a heterogenic response in endothelial cells with regard to VWF expression, leading to organ-specific increase in VWF levels and alterations in vascular tree pattern of expression. This is concomitant with increased platelet aggregate formation. The age-associated increase in VWF expression may be modulated through the process of cell senescence, and p53 transcription factor contributes to its regulation.

Telomeres shorten with each cell division, acting as a chronometer of cell age. The enzyme telomerase, primarily active in stem cells, reverses telomere erosion. We have previously observed that transient transfection with human TERT mRNA extends telomeres and mitigates hallmarks of senescence in replicatively aged human cells or those affected by Hutchinson–Gilford progeroid syndrome (HGPS). However, due to its short half‐life, mRNA requires frequent administration. In this study, we hypothesized that TERT circular (circ) RNA would extend the duration of telomerase expression and be more effective at reversing hallmarks of senescence in endothelial cells derived from HGPS patients. We observe that a single transfection of TERT circRNA is more effective than mRNA in the extension of telomere length, as determined by quantitative fluorescence in situ hybridization. Furthermore, TERT circRNA reduced the number of β‐gal positive cells by three‐fold and normalized nuclear morphology in HGPS endothelial cells (HGPS‐ECs). Moreover, TERT circRNA substantially reduced senescent markers, inflammatory markers, and DNA damage markers, including Progerin, p16, p21, IL‐1B, IL‐6, IL‐8, MCP1, and γH2AX. Additionally, it restored NO production, enhanced cell proliferation, promoted angiogenesis, improved LDL uptake, reduced mitochondrial ROS, and normalized mitochondrial membrane potential more effectively. Our data suggest that TERT circRNA is superior to linear TERT mRNA in reversing processes involved in senescence.

Cell-penetrating peptides (CPPs) are effective for delivering therapeutic molecules with minimal toxicity. This study focuses on the use of penetratin, a well-characterized CPP, to deliver a DNA vector encoding short hairpin RNA (shRNA) targeting the respiratory syncytial virus (RSV) F gene into infected cells. RSV is known to cause severe lower respiratory infections in infants and poses significant risks to immunocompromised individuals and the elderly. We evaluated the antiviral efficacy of the penetratin-shRNA complex by comparing its ability to inhibit RSV replication and induce apoptosis with ribavirin treatment. Penetratin-shRNA complexes were prepared at different ratios and analyzed using gel retardation assays, dynamic light scattering, and zeta potential measurements. The complexes were tested in HEp-2 and A549 cells for transfection efficiency, cytotoxicity, viral load, and apoptosis using plaque assays, real-time reverse transcription-polymerase chain reaction (RT-PCR), DNA fragmentation, propidium iodide staining, and caspase 3/7 activation assays. The gel shift assay determined that a 20:1 CPP-to-shRNA ratio was optimal for effective complexation, resulting in particles with a size of 164 nm and a zeta potential of 8.7 mV. Transfection efficiency in HEp-2 cells was highest at this ratio, reaching up to 93%. The penetratin-shRNA complex effectively silenced the RSV F gene, reduced viral titers, and decreased DNA fragmentation and apoptosis in infected cells. Penetratin effectively delivers shRNA targeting the RSV F gene, significantly reducing viral load and preventing apoptosis without toxicity. This approach surpasses Lipofectamine and shows potential for future therapeutic interventions, especially when combined with ribavirin, against RSV infection.

Radiation therapy, while effective against cancer, often causes collateral damage to surrounding healthy tissues, leading to DNA damage that can precipitate genomic instability and cancer. Despite the enormity of the problem, there is currently no FDA-approved agent to prevent or treat skin damage caused by ionizing radiation. In the current study, ionizing radiation induced dose-dependent genomic and mitochondrial DNA damage, leading to apoptosis in primary cutaneous cells. Prior treatment with mRNA encoding telomerase reverse transcriptase (TERT) substantially reduced radiation induced DNA damage in human primary skin cells and tissues. Mechanistically, TERT mRNA pretreatment enhances DNA repair through the homologous recombination pathway, reduces mitochondrial ROS, and decreases apoptosis without extending telomere length during the experimental period, suggesting a non-canonical function of TERT to accelerate cellular recovery from radiation. These findings highlight a potential therapeutic approach for preventing radiation-induced skin injury.

The transduction of external forces to the internal components of the cell has critical implications for cell behavior in health and disease. Despite advances in techniques such as atomic force microscopy (AFM), accurately and reproducibly estimating the mechanical properties of multiple individual subcellular compartments remains challenging. This is often due to variability in measurements, the lack of a standard "inverse" approach to estimate unknown properties, and an often ill-posed inverse problem. This study presents an integrated experimental-computational framework for the optimal design of an inverse approach to estimate multi-compartment cell properties, focusing on the behavior of the nuclear membrane, cytoplasm, and nucleoplasm. The optimal design approach identifies an ideal set of AFM measurements that minimizes the dependence of the estimated multi-compartment properties on AFM probing locations, thereby improving the reproducibility of mechanical property estimations. Super-resolution imaging was used to construct a 3-D computational model of a human umbilical vein endothelial cell (HUVEC). An inverse modeling approach was then used to fit experimental data to a hyperelastic constitutive model, accounting for large-deformation nonlinearities. Simulations incorporating visco-hyperelasticity were conducted to explore viscous effects. Our approach leads to quantification of the mechanical properties of the nucleoplasm, nuclear membrane, and cytoplasm with minimal dependence on loading conditions. We expect our approach to assist with standardizing the biomechanical characterizations of subcellular structures, improve the consistency and reproducibility of the estimations across mechanobiological studies, and ultimately improve our understanding of the role of mechanotransduction in disease progression.