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The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify motion of the peripheral membrane of cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, stiffness consistent with a healthy pulmonary artery. Unlike these large length scale morphological changes, the baseline of PMFs was largely insensitive to substrate stiffness on which cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel - a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate: (i) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; (ii) PMFs increase in response to calcium influx through store-operated calcium entry channels; and (iii) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

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Patients who recover from nosocomial pneumonia oftentimes exhibit long‐lasting cognitive impairment comparable to what is observed in Alzheimer’s Disease patients. We previously hypothesized that the lung endothelium contributes to infection‐related neurocognitive dysfunction, since bacteria‐exposed endothelial cells release a form(s) of cytotoxic tau that is sufficient to impair long‐term potentiation in the hippocampus. However, lung endothelial tau isoform(s) have yet to be resolved and it remains unclear whether the infection‐induced endothelial cytotoxic tau can trigger neuronal tau aggregation which is the major hallmark of several neuropathologies. Here, we demonstrate that lung endothelial cells express a big tau isoform and three additional tau isoforms that are similar to neuronal tau, each containing four microtubule‐binding repeat domains, and that tau is expressed in lung capillaries in vivo. To test whether infection elicits endothelial tau capable of causing transmissible tau aggregation, cells were infected with P. aeruginosa. The infection‐induced tau released from endothelium into the medium induced neuronal tau aggregation in reporter cells, including reporter cells that express either the four microtubule‐binding repeat domains or the full‐length tau. Infection‐induced release of pathological tau variant(s) from endothelium, and the ability of the endothelial‐derived tau to cause neuronal tau aggregation, was abolished using tau knockout cells. After bacterial lung infection, brain homogenates from wild‐type mice, but not from tau knockout mice, initiated tau aggregation. Notably, plasma samples obtained from pneumonia‐positive patients showed significantly higher tau aggregation activity when compared to pneumonia‐negative plasma samples. Thus, bacterial pneumonia initiates the release of lung endothelial‐derived cytotoxic tau into the circulation, which is capable of propagating a neuronal tauopathy.

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