Gabriel Kelly Abstracts

Gabriel Kelly

Ph.D. Candidate

Physiological Sciences GIDP

42nd Annual Conference on Shock

Coronado, California

June 8-11, 2019

Introduction: Treatment for critically ill patients of acute respiratory distress syndrome (ARDS), sepsis, and other pulmonary injuries often includes mechanical ventilation (MV). Though necessary and often lifesaving, MV exposes the lung to further stress, distension, and unnatural pressures, leading to ventilator induced lung injury (VILI). One hallmark of VILI includes loss of endothelial barrier integrity, leading to influx of immune cells, cytokines, and edema into the alveolar space. We have recently found that integrin β4 (ITGβ4 or CD104, gene code: ITGB4), a transmembrane receptor protein also involved in cellular signaling and cell-matrix communication, plays a key role in endothelial barrier regulation during VILI. Multiple mRNA splice variants of the ITGB4 gene exist, including the E variant (ITGβ4E), the only variant which has a unique 114 amino acid cytoplasmic domain, compared to the full length ITGβ4C. Our recent findings suggest that ITGβ4E may be a key protective ITGB4 splicing variant against VILI.

Methods: We exposed human lung endothelial cells (ECs) to simvastatin and TNFα and evaluated ITGβ4E expression patterns using RT-PCR and western blotting. Simvastatin treated cells were also imaged using immunohistochemistry. Additionally, ECs were infected with adenovirus containing ITGβ4C or E expression vectors and subjected to electric cell-substrate impedance sensing (ECIS) to measure integrity of the monolayer in response to thrombin.

Results: Simvastatin upregulated all mRNA variants of the ITGB4 gene, with an initial spike in ITGβ4E variant expression. Confocal imaging results confirmed that simvastatin upregulates ITGβ4, with differential cellular distributions of ITGβ4E compared with other forms of ITGβ4. Using an antibody specific for the extracellular domain that recognizes all variants of ITGβ4, we observed a uniform distribution of ITGβ4, with apparent cell surface clustering in about 50% of cells. Co-staining studies using an antibody that recognizes only ITGβ4E indicate that ITGβ4E does not participate in clustering, and has a more centralized distribution as compared to ITGβ4. TNFα treatment resulted in an initial drop in ITGβ4E mRNA, followed by a general drop in all ITGβ4 mRNA. ECs subjected to thrombin resulted in a drop in monolayer impedance, but ITGβ4E infected cells were partially protected compared to ITGβ4C infected cells. Additionally, ITGβ4E infected ECs recovered more quickly than control while ITGβ4C infected EC recovery was not significant.

Conclusions: Our results indicate that exposure to simvastatin and TNFα influence ITGB4 transcription and splicing of ITGβ4, with variable distribution patterns between variants, suggesting alternative functions. ITGβ4E seems to offer more protection than ITGβ4C in monolayers subjected to chemical insults. Current work is underway to understand differing signaling capabilities of ITGβ4 variants.

This study is supported in part by National Institutes of Health grants T32HL007249, HL134610, and P01HL126609.

Abstract for Lay Audience

Some of the most dangerous results of acute respiratory distress syndrome (ARDS) and sepsis are an inability to breathe and provide oxygen to the blood. Patients of these diseases suffer from a very high death rate and there are no accepted therapeutics. Patients who experience these diseases are often put on ventilators to help them breathe, however, mechanical ventilation leads to more damage to the lung because of the unnatural pressures of the system. This leads to ventilator induced lung injury (VILI). All of these problems include a weakening of the barrier between the lung air space and the blood, allowing fluid and other harmful chemicals to enter the lung. Our research focusses on proteins that determine the shape and strength of cells that line the blood vessels (endothelial cells). We have recently found that one protein, integrin beta4 (ITGB4), which is one protein that anchors endothelial cells to the tissue around it, regulates the endothelial barrier strength during VILI. Specifically, one variant of this protein, ITGB4E, has shown extremely protective effects in mice and cell models.

We used human endothelial cells to analyze ITGB4E’s function in response to various chemicals. Simvastatin is known to strengthen the barrier, while TNFa and thrombin are known to damage the barrier. We used basic molecular techniques to measure ITGB4E levels, image cellular locations, and measure barrier strength.

The protective chemical simvastatin increased ITGB4E levels more than other variants of this protein, and ITGB4E was seen to locate closer to the bottom surface of cells. Other variants were also upregulated, though not as much as E, and were seen at locations where cells touch neighboring cells. TNFa reduced ITGB4 levels, and especially reduced ITGB4E. When cells were treated with thrombin, the strength of the barrier was weakened, but cells with extra ITGB4E were protected.

Our results show that ITGB4E behaves differently than other variants of this protein and offers protection in models of lung injury. Thus ITGB4 levels may be a potential target for ARDS and VILI therapeutics. Understanding how endothelial cells behave during injury and protection will help us understand the course of these diseases going forward