integrative-toolkit-to-analyze-cellular-signals-forces-motion

Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Quantitative assessment of cellular forces and motion advanced considerably over the last four decades. These advancements provided the framework to ...

University of South Alabama

It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier-disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier-protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that, within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4-sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of acute respiratory distress syndrome patients. 

Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10.

Studies on the resolution of subcellular free calcium concentrations: a technological advance. Focus on "detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells".

Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membrane-associated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by ∼50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1. 

Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1.

The canonical transient receptor potential channel 4 (TRPC4) comprises an endothelial store-operated Ca(2+) entry channel, and TRPC4 inactivation confers a survival benefit in pulmonary arterial hypertension (PAH). Endothelial Ca(2+) signals mediated by TRPC4 enhance vascular permeability in vitro, but the contribution of TRPC4-dependent Ca(2+) signals to the regulation of endothelial permeability in PAH is poorly understood. We tested the hypothesis that TRPC4 increases vascular permeability and alters the frequency of endothelial Ca(2+) transients in PAH. We measured permeability in isolated lungs, and found that TRPC4 exaggerated permeability responses to thapsigargin in Sugen/hypoxia-treated PAH rats. We compared endothelial Ca(2+) activity of wild-type with TRPC4-knockout rats using confocal microscopy, and evaluated how Ca(2+) signals were influenced in response to thapsigargin and sequential treatment with acetylcholine. We found that thapsigargin-stimulated Ca(2+) signals were increased in PAH, and recovered by TRPC4 inactivation. Store depletion revealed bimodal Ca(2+) responses to acetylcholine, with both short- and long-duration populations. Our results show that TRPC4 underlies an exaggerated endothelial permeability response in PAH. Furthermore, TRPC4 increased the frequency of endothelial Ca(2+) transients in severe PAH, suggesting that TRPC4 provides a Ca(2+) source associated with endothelial dysfunction in the pathophysiology of PAH. This phenomenon represents a new facet of the etiology of PAH, and may contribute to PAH vasculopathy by enabling inflammatory mediator flux across the endothelial barrier.

Transient Receptor Potential Channel 4 Encodes a Vascular Permeability Defect and High-Frequency Ca(2+) Transients in Severe Pulmonary Arterial Hypertension.

The canonical transient receptor potential 4 (TRPC4) protein contributes to the molecular make-up of endothelial store-operated calcium entry channels. Store-operated calcium entry is a prominent mode of calcium influx in endothelium. Store-operated calcium entry channels are activated by inflammatory mediators and growth factors, and in endothelium, this process induces inter-endothelial cell gaps that increase permeability. Pulmonary endothelium within extra-alveolar segments, including pulmonary arteries, is especially sensitive to the activation of store-operated calcium entry. Pulmonary arterial hypertension (PAH) is characterized by endothelial cell dysfunction in arteries. As one of the topics for the 2017 Grover Conference Series, we examined whether an endothelial cell permeability defect accompanies PAH and, if so, whether TRPC4 contributes to this defect. Through a series of studies conducted over the past five years, we find endothelial cell barrier dysfunction occurs early in the progression of experimental PAH. Endothelium within the arterial segment, and perhaps in other vascular segments, is highly susceptible to disruption secondary to both activation of store-operated calcium entry channels and high flow. This phenomenon partly depends upon TRPC4 channels. We discuss whether endothelial cell hyperpermeability is relevant to human disease, and more specifically, whether it is relevant to all groups of pulmonary hypertension.

The role of endothelial leak in pulmonary hypertension (2017 Grover Conference Series).

The mechanical microenvironment of an endothelial cell includes a stable protein scaffold on the basal side, flowing blood on the apical side and contractile cells on the lateral sides. Interaction with the protein scaffold and flowing blood modulates the ability of endothelial cells to migrate, align and maintain barrier function. Interaction with neighbors provides the endothelial monolayer unique "collective" properties. However, the nature of local mechanical signaling - i.e., the local functional consequence of a cell interacting with its contractile neighbors - remains unclear. Using an advancing sheet of pulmonary microvascular endothelial cells, here we examine the mechanical properties of an individual cell and its neighboring region. By combining Monolayer Stress Microscopy (MSM) with a novel analysis, we assessed several mechanical properties of an individual cell and its neighboring region. Across the monolayer, mechanical properties of the neighboring region defined multicellular "subdivisions" wherein constituent cells were exposed to a similar mechanical microenvironment. Adjacent subdivisions were separated by a narrow interface where adjoining cells were exposed to remarkably different mechanical microenvironments. Comparison of temporal fluctuations in mechanical properties of individual cells and those of their neighboring regions suggested three distinct intercellular mechanical signaling processes. These processes indicated that change in size, shape and speed of individual cells is associated with change in contractile forces in their neighboring regions. In summary, we present a novel approach to assess the mechanical interactions of individual cells with their contractile neighbors and identify potential functional consequences of such interactions.

Mechanical signaling in a pulmonary microvascular endothelial cell monolayer.

In the field of endothelial biology, the term "shear forces" is tied to the forces exerted by the flowing blood on the quiescent cells. But endothelial cells themselves also exert physical forces on their immediate and distant neighbors. Specific factors of such intrinsic mechanical signals most relevant to immediate neighbors include normal (F) and shear (F) components of intercellular tractions, and those factors most relevant to distant neighbors include contractile or dilatational (M) and shear (M) components of the moments of cytoskeletal forces. However, for cells within a monolayer, F, F, M, and M remain inaccessible to experimental evaluation. Here, we present an approach that enables quantitative assessment of these properties. Remarkably, across a collectively migrating sheet of pulmonary microvascular endothelial cells, F was of the same order of magnitude as F. Moreover, compared to the normal components (F, M) of the mechanical signals, the shear components (F, M) were more distinctive in the cells closer to the migration front. Individual cells had an innately collective tendency to migrate along the axis of maximum contractile moment - a collective migratory process we referred to as cellular plithotaxis. Notably, larger F and M were associated with stronger plithotaxis, but dilatational moment appeared to disengage plithotactic guidance. Overall, cellular plithotaxis was more strongly associated with the "shear forces" (F, M) than with the "normal forces" (F, M). Finally, the mechanical state of the cells with fast migration speed and those with highly circular shape were reminiscent of fluid-like and solid-like matter, respectively. The results repeatedly pointed to neighbors imposing shear forces on a cell as a highly significant event, and hence, the term "shear forces" must include not just the forces from flowing fluid but also the forces from the substrate and neighbors. Collectively, these advances set the stage for deeper understanding of mechanical signaling in cellular monolayers.

Unleashing shear: Role of intercellular traction and cellular moments in collective cell migration.

Collective migration of endothelial cells is important for wound healing and angiogenesis. During such migration, each constituent endothelial cell coordinates its magnitude and direction of migration with its neighbors while retaining intercellular adhesion. Ensuring coordination and cohesion involves a variety of intra- and inter-cellular signaling processes. However, the role of permeation of extracellular Na+ in collective cell migration remains unclear. Here, we examined the effect of Na+ permeation in collective migration of pulmonary artery endothelial cell (PAEC) monolayers triggered by either a scratch injury or a barrier removal over 24 hours. In the scratch assay, PAEC monolayers migrated in two approximately linear phases. In the first phase, wound closure started with fast speed which then rapidly reduced within 5 hours after scratching. In the second phase, wound closure maintained at slow and stable speed from 6 to 24 hours. In the absence of extracellular Na+, the wound closure distance was reduced by >50%. Fewer cells at the leading edge protruded prominent lamellipodia. Beside transient gaps, some sustained interendothelial gaps also formed and progressively increased in size over time, and some fused with adjacent gaps. In the absence of both Na+ and scratch injury, PAEC monolayer migrated even more slowly, and interendothelial gaps obviously increased in size towards the end. Pharmacological inhibition of the epithelial Na+ channel (ENaC) using amiloride reduced wound closure distance by 30%. Inhibition of both the ENaC and the Na+/Ca2+ exchanger (NCX) using benzamil further reduced wound closure distance in the second phase and caused accumulation of floating particles in the media. Surprisingly, pharmacological inhibition of the Ca2+ release-activated Ca2+ (CRAC) channel protein 1 (Orai1) using GSK-7975A, the transient receptor potential channel protein 1 and 4 (TRPC1/4) using Pico145, or both Orai1 and TRPC1/4 using combined GSK-7975A and Pico145 treatment did not affect wound closure distance dramatically. Nevertheless, the combined treatment appeared to cause accumulation of floating particles. Note that GSK-7975A also inhibits small inward Ca2+ currents via Orai2 and Orai3 channels, whereas Pico145 also blocks TRPC4, TRPC5, and TRPC1/5 channels. By contrast, gene silence of Orai1 by shRNAs led to a 25% reduction of wound closure in the first 6 hours but had no effect afterwards. However, in the absence of extracellular Na+ or cellular injury, Orai1 did not affect PAEC collective migration. Overall, the data reveal that Na+ permeation into cells contributes to PAEC monolayer collective migration by increasing lamellipodial formation, reducing accumulation of floating particles, and improving intercellular adhesion.

Article	Year
Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 23686854	2013
Studies on the resolution of subcellular free calcium concentrations: a technological advance. Focus on "detection of differentially regulated subsarcolemmal calcium signals activated by vas... American journal of physiology. Cell physiology\| PubMed ID: 24553184	2014
Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1. American journal of physiology. Cell physiology\| PubMed ID: 25428882	2015
Transient Receptor Potential Channel 4 Encodes a Vascular Permeability Defect and High-Frequency Ca(2+) Transients in Severe Pulmonary Arterial Hypertension. The American journal of pathology\| PubMed ID: 27083517	2016
The role of endothelial leak in pulmonary hypertension (2017 Grover Conference Series). Pulmonary circulation\| PubMed ID: 30124139	2018
Mechanical signaling in a pulmonary microvascular endothelial cell monolayer. Biochemical and biophysical research communications\| PubMed ID: 31514994	2019
Unleashing shear: Role of intercellular traction and cellular moments in collective cell migration. Biochemical and biophysical research communications\| PubMed ID: 31879014	2020
Impact of Na+ permeation on collective migration of pulmonary arterial endothelial cells. PloS one\| PubMed ID: 33891591	2021

Ningyong Xu

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