In response to stress in the environment, many organisms respond by accumulating molecularly small solutes termed osmolytes. Curiously, two osmolytes, TMAO and urea, act together in elasmobranchs such as sharks to form solutions at molar concentrations whose net action is close to that of pure water. Although it is known that many osmolytes exert an apparent attractive or repulsive force between self-assembled lipid membranes, all proposed models fail to fully account for the origin of this force. Toward resolving the mechanism by which osmolytes modulate lipid interactions, we followed several osmolytes, including urea and TMAO, and their interaction with lipid membranes in aqueous solution. [1] We found that TMAO pushes adjacent membranes closer together, while urea makes membranes swell. Experiments and simulations further show that the change in the force between membranes is due to the partitioning of TMAO away from the volume between bilayers. This in turn stems from the exclusion of TMAO from the lipid-water interface. Hence, the underlying mechanism resembles protein stabilization by osmolytes. By contrast urea is almost equally partitioned in lipid and bulk, and its action is mostly related to modified van der Waals interactions. Interestingly, urea and TMAO act synergistically, so that the presence of one changes the preferential interaction of the other with lipids. We discuss the potential role of osmolytes acting together in the modifications of lipid adhesion and fusion processes. References [1] S.
Endothelial cells (ECs) are in the inner layer of blood vessels, and it controls the transportation of nutrition and essential molecules from the extracellular matrix to the intracellular space making a homeostasis environment. Vascular endothelium can be disrupted with viruses, which are among the main reasons for epidemic and pandemic outbreaks, and it leads to health issues such as cardiovascular‐related diseases. Wide range of viruses can potentially target the endothelium including Dengue virus estimated to infect 390 million people per year. Zika virus leads to pregnancy complications such as preterm birth and miscarriage. Tick‐borne encephalic damaged the central nervous system and, Hantavirus has a mortality rate of 38% through hantavirus pulmonary syndrome. Reviewing these several diseases, including SARS‐CoV‐2, has given an in‐depth look at what the endothelial cells are going through when these viruses are infecting the host bodies. Following the virus infection, ECs are going through activation mode, which is accompanied with glycocalyx degradation, plasma leakage, phosphorylation of P120, and cadherin, increased proinflammatory responses and, disruption of barrier integrity. The SARS‐CoV‐2 pandemic has taken a global toll; With a total of 362 million cases, there are 5.63 million deaths occurring. With the usage of immunofluorescence and vascular permeability assay, we monitor how Human Umbilical Vascular Endothelial Cells (HUVEC) and Human Lung Microvascular Endothelial Cells (HLMVEC) are impacted when treated with UV‐inactivated & heat‐inactivated SARS‐CoV‐2, and tumor necrosis factor (TNF)‐α. TNF‐α is an inflammatory cytokine prominent during inflammation and its known to increase the permeability of endothelium. Like the TNF‐α, both cells treated with UV‐inactivated and heat‐inactivated SARS‐CoV2 demonstrated boosted permeability. With the considerable effect of SARS‐CoV2 on the permeability of ECs, it is essential to understand the mechanisms and pathways behind the boosted leak, including the impact of the virus on the expression of adhesion molecules like platelet endothelial cell adhesion molecule (PECAM‐1). One can see in the images of HUVEC and HLMVEC that SARS‐CoV‐2 diminish the localization of PECAM‐1. On a non‐treated negative control, cells are spaced evenly and are close to another since their junctions are not targeted. Once the cells are treated with either heat‐inactivated or UV‐inactivated SARS‐CoV‐2, PECAM‐1 localization is disrupted, which can be seen from the space between cells since the PECAM‐1 has been altered and is no longer holding the cells together. From those exact images, one can detect the enlarged and irregular shapes of nucleuses and change in morphology of these cells since the SARS‐CoV‐2 is not only altering the PECAM‐1 complex but it can also modify various proteins and mRNAs. By combining information of various viruses and knowledge from our studies, we can understand the mechanisms of ECs and take measurements to protect ECs from viruses. In future experiments,...
Endothelial surface glycocalyx (ESG) is a carbohydrate‐rich layer found on the endothelial cell (EC) surface lining all blood vessels' inner lumen. Composed of membrane glycoproteins, glycosaminoglycans (GAGs), such as hyaluronan and heparan sulfate (HS), and proteoglycans, the ESG forms a bulky, gel‐like layer serving critical functions in blood flow mechanotransduction, endothelial permeability maintenance, leukocyte adhesion, and inflammation control. ESG is known for sensing shear stress of blood flow and transducing the mechanical signal into nitric oxide (NO) production. NO is an essential signaling molecule to regulate vascular tone. To date, the molecular pathways of ESG‐mediated mechanotransduction have not been completely clear. We utilize a custom‐built AFM with fluorescence imaging capabilities to vertically stretch the ESG, which applies a pulling force to specific ESG components on the surface of a single live mouse brain endothelial cell (bEnd.3) and quantify the cell's NO production in real‐time. NO production is quantified by the fluorescent dye DAF‐FM. AFM probe is coated with monoclonal antibodies against HS or glypican‐1 (a major core protein for HS) to exert pulling forces onto the HS or glypican‐1 on bEnd.3. To ensure that DAF intensity is a signal of NO production, we treated the cells with L‐NAME (an inhibitor of endothelial nitric oxide synthase). The DAF intensity was greatly reduced, which confirms that the DAF intensity is representing NO production. The AFM anti‐glypican‐1 coated probe showed an increase in DAF intensity, while the anti‐HS probe is shown to have a slight rise in DAF intensity. Since HS may attach to other proteins besides glypican, the data suggest that glypican‐1 is a more critical mechanotransducer. Our prior study indicated cation‐permeable, transient receptor potential (TRP) ion channels mediate stretch‐induced NO production. Probes were coated in glypican‐1 while the cells were exposed to Amiloride or SKF96365, blockers of TRPP2 and TRPC1 channels, respectively. After the Amiloride‐treated cells were mechanically stimulated, there was a reduction of NO production. However, the amount of NO output was still significant over the basal levels observed in cells. When the SKF96365‐treated cells were mechanically stimulated, production was inhibited entirely, thus, demonstrating TRPC1 channels play a vital role in allowing the Ca2+ into the cell to begin NO production cascade. These findings confirm the critical roles of HS, glypican‐1, and TRP channels on the rapid NO production resulting from mechanical signaling and have important implications for ESG‐related cardiovascular diseases such as diabetes, strokes, cardiac arrest, and atherosclerosis. It has been shown that intracellular Ca2+regulates eNOS activity. In future studies, we will measure the Ca2+intake mediated by TRP channels and delineate the interplay among mechanical stretch, TRP channel activation, Ca2+intake and NO production.
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