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Dendritic cells (DCs), professional antigen-presenting cells, function as sentinels of the immune system. DCs initiate and fine-tune adaptive immune responses by presenting antigenic peptides to B and T lymphocytes to mount an effective immune response against cancer and pathogens. However, hypoxia, a condition characterized by low oxygen (O2-cryogels) tension in different tissues, significantly impacts DC functions, including antigen uptake, activation, and maturation, migration, as well as T-cell priming and proliferation. In this study, we employed O2-releasing biomaterials (O2-cryogels) to study the effect of localized O2 supply on human DC phenotype and functions. Our results indicate that O2-cryogels effectively mitigate DC exposure to hypoxia under hypoxic conditions. Additionally, O2--cryogels counteract hypoxia-induced inhibition of antigen uptake and migratory activity in DCs through O2-release and hyaluronic acid (HA) mediated mechanisms. Furthermore, O2-cryogels preserve and restore DC maturation and co-stimulation markers, including HLA-DR, CD86, and CD40, along with the secretion of proinflammatory cytokines in hypoxic conditions. Finally, our findings demonstrate that the supplemental O2-released from the cryogels preserves DC-mediated T-cell priming, ultimately leading to the activation and proliferation of allogeneic CD3+ T cells. This work emphasizes the potential of local oxygenation as a powerful immunomodulatory agent to improve DC activation and functions in hypoxia, offering new approaches for cancer and infectious disease treatments. 

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies. 

The genetic material within cells plays a pivotal role in shaping the structure and function of living organisms. Manipulating an organism's genome to correct inherited abnormalities or introduce new traits holds great promise. Genetic engineering techniques offers promising pathways for precisely altering cellular genetics. Among these methodologies, clustered regularly interspaced short palindromic repeat (CRISPR), honored with the 2020 Nobel Prize in Chemistry, has garnered significant attention for its precision in editing genomes. However, the CRISPR system faces challenges when applied in vivo, including low delivery efficiency, off‐target effects, and instability. To address these challenges, innovative technologies for targeted and precise delivery of CRISPR have emerged. Engineered carrier platforms represent a substantial advancement, improving stability, precision, and reducing the side effects associated with genome editing. These platforms facilitate efficient local and systemic genome engineering of various tissues and cells, including immune cells. This review explores recent advances, benefits, and challenges of CRISPR‐based genome editing delivery. It examines various carriers including nanocarriers (polymeric, lipid‐derived, metallic, and bionanoparticles), viral particles, virus‐like particles, and exosomes, providing insights into their clinical utility and future prospects. 

Glycocalyx (GCX) is a carbohydrate-rich structure that coats the surface of endothelial cells (ECs) and lines the blood vessel lumen. Mechanical perturbations in the vascular environment, such as blood vessel stiffness, can be transduced and sent to ECs through mechanosensors such as GCX. Adverse stiffness alters GCX-mediated mechanotransduction and leads to EC dysfunction and eventually atherosclerotic cardiovascular diseases. To understand GCX-regulated mechanotransduction events, anin vitromodel emulatingin vivovessel conditions is needed. To this end, we investigated the impact of matrix chemical and mechanical properties on GCX expression via fabricating a tunable non-swelling matrix based on the collagen-derived polypeptide, gelatin. To study the effect of matrix composition, we conducted a comparative analysis of GCX expression using different concentrations (60–25,000 μg/mL) of gelatin and gelatin methacrylate (GelMA) in comparison to fibronectin (60 μg/mL), a standard coating material for GCX-related studies. Using immunocytochemistry analysis, we showed for the first time that different substrate compositions and concentrations altered the overall GCX expression on human umbilical vein ECs (HUVECs). Subsequently, GelMA hydrogels were fabricated with stiffnesses of 2.5 and 5 kPa, representing healthy vessel tissues, and 10 kPa, corresponding to diseased vessel tissues. Immunocytochemistry analysis showed that on hydrogels with different levels of stiffness, the GCX expression in HUVECs remained unchanged, while its major polysaccharide components exhibited dysregulation in distinct patterns. For example, there was a significant decrease in heparan sulfate expression on pathological substrates (10 kPa), while sialic acid expression increased with increased matrix stiffness. This study suggests the specific mechanisms through which GCX may influence ECs in modulating barrier function, immune cell adhesion, and mechanotransduction function under distinct chemical and mechanical conditions of both healthy and diseased substrates. 

Endothelium health is essential to the regulation of physiological vascular functions. Because of the critical capability of endothelial cells (ECs) to sense and transduce chemical and mechanical signals in the local vascular environment, their dysfunction is associated with a vast variety of vascular diseases and injuries, especially atherosclerosis and subsequent cardiovascular diseases. This review describes the mechanotransduction events that are mediated through ECs, the EC subcellular components involved, and the pathways reported to be potentially involved. Up-to-date research efforts involving in vivo animal models and in vitro biomimetic models are also discussed, including their advantages and drawbacks, with recommendations on future modeling approaches to aid the development of novel therapies targeting atherosclerosis and related cardiovascular diseases.