Calcium-dependent cAMP mediates the mechanoresponsive behaviour of endothelial cells to high-frequency nanomechanostimulation.

The endothelial junction plays a central role in regulating intravascular and interstitial tissue permeability. The ability to manipulate its integrity therefore not only facilitates an improved understanding of its underlying molecular mechanisms but also provides insight into potential therapeutic solutions. Herein, we explore the effects of short-duration nanometer-amplitude MHz-order mechanostimulation on interendothelial junction stability and hence the barrier capacity of endothelial monolayers. Following an initial transient in which the endothelial barrier is permeabilised due to Rho-ROCK-activated actin stress fibre formation and junction disruption typical of a cell's response to insults, we observe, quite uniquely, the integrity of the endothelial barrier to not only spontaneously recover but also to be enhanced considerably-without the need for additional stimuli or intervention. Central to this peculiar biphasic response, which has not been observed with other stimuli to date, is the role of second messenger calcium and cyclic adenosine monophosphate (cAMP) signalling. We show that intracellular Ca2+, modulated by the high frequency excitation, is responsible for activating reorganisation of the actin cytoskeleton in the barrier recovery phase, in which circumferential actin bundles are formed to stabilise the adherens junctions via a cAMP-mediated Epac1-Rap1 pathway. Despite the short-duration stimulation (8 min), the approximate 4-fold enhancement in the transendothelial electrical resistance (TEER) of endothelial cells from different tissue sources, and the corresponding reduction in paracellular permeability, was found to persist over hours. The effect can further be extended through multiple treatments without resulting in hyperpermeabilisation of the barrier, as found with prolonged use of chemical stimuli, through which only 1.1- to 1.2-fold improvement in TEER has been reported. Such an ability to regulate and enhance endothelial barrier capacity is particularly useful in the development of in vitro barrier models that more closely resemble their in vivo counterparts.

In vivo deposition study of a new generation nebuliser utilising hybrid resonant acoustic (HYDRA) technology.

Conventional nebulisation has the disadvantages of low aerosol output rate and potential damage to macromolecules due to high shear (jet nebulisation) or cavitation (ultrasonic nebulisation). HYDRA (HYbriD Resonant Acoustics) technology has been shown to overcome these problems by using a hybrid combination of surface and bulk sound waves to generate the aerosol droplets. We report the first in vivo human lung deposition study on such droplets. Twelve healthy adult subjects inhaled saline aerosols radiolabelled with technetium-99 m complexed with diethylene triamine penta-acetic acid (99mTc-DTPA). The distribution of the aerosolised droplets in the lungs was imaged by single photon emission computed tomography combined with low dose computed tomography (SPECT/CT). The volume median diameter and geometric standard deviation of the droplets were 1.32 ± 0.027 µm and 2.06 ± 0.040, respectively. The mean delivery efficiency from the nebuliser into the body was 51.2%. About 89.1 ± 4.3% and 2.3 ± 1.4% of the inhaled radiolabelled dose deposited in the lungs and oropharynx, respectively. The deposition was symmetrical and diffusive between the two lungs, with a mean penetration index of 0.82. Thus, the prototype HYDRA nebuliser showed excellent in vivo aerosol deposition performance, demonstrating its potential to be further developed for clinical applications.