Understanding resilience — the ability of injured lung tissue to heal and regenerate — may be key to advancing the treatment and prevention of a life-threatening lung disease that occurs in extremely premature babies, a new study suggests.
Using a four-dimensional microscopy technique, researchers at Vanderbilt University and Vanderbilt University Medical Center have created 3D video images of mouse lung tissue grown in the laboratory. What they have learned has been nothing short of groundbreaking.
“For the first time, we’ve been able to live-image the lung as it forms, and quantify and measure those cellular movements that come together to make an organ with a surface area large enough for gas exchange,” said Jennifer Sucre, MD, associate professor of Pediatrics and Cell and Developmental Biology.
The group’s findings, published in print Feb. 24 by the American Society for Clinical Investigation in its journal, JCI Insight, represent a significant step toward improved treatment and prevention of bronchopulmonary dysplasia (BPD), which occurs in about 50% of infants born two to four months prematurely.
“If we can understand how the lung forms, then we have a blueprint for how to grow new lungs after injury,” said the paper’s first author, Nick Negretti, PhD, a senior post-doctoral fellow in the Sucre lab who co-led the research.
“Mice have an extraordinary ability to repair the lung,” said Sucre, the paper’s senior author, who directs the Biodevelopmental Origins of Lung Disease (BOLD) Center at VUMC. “I want to give babies the superpower of the mouse.”
Premature babies with BPD require oxygen and mechanical ventilation in the early days after birth to help them breathe. Oxygen therapy is a double-edged sword, however, because it also can damage delicate lung tissue.
Though many premature babies can be weaned off the ventilator after a few days, they are at increased risk for developing serious breathing problems later in life, including chronic obstructive pulmonary disease.
Respiration — the exchange of oxygen for carbon dioxide — occurs in the alveoli of the lungs across a fragile basement membrane between epithelial cells and blood vessels. According to the traditional view of lung development, ingrowing septa (dividers) emerge from a layer of epithelial, endothelial and mesenchymal cells to divide airspaces into the alveoli.
But when the researchers imaged slices of living neonatal mouse lung over three days using a technique called scanned oblique plane illumination (SOPi) microscopy, a different view emerged, one of a ballooning outgrowth of epithelial cells supported by a ring of myofibroblasts, or cells that promote tissue formation.
“In more than 2,000 hours of live imaging, we never saw a single instance of ingrowing septation,” Negretti said. “The structures that we previously thought were ingrowing septal tips were artifacts of traditional two-dimensional imaging.”
Development of the alveoli (alveologenesis) is a precisely timed and coordinated set of molecular cues delivered by myofibroblasts via intercellular signaling pathways and transmitted mechanical forces. The innovative technology implemented by the Sucre lab allows for testing and identification of the specific molecules and pathways that guide this process.
As proof of principle that their 4D system and computational analysis quantitatively modelled both normal and perturbed tissue development, the researchers showed that small-molecule modulators of Wnt intercellular signaling pathways and mechanical cellular processes inhibited epithelial cell movement and differentiation.
“Getting the model right and understanding how the cells move changes the questions we’re asking,” Sucre said. “Instead of looking for cells that divide alveoli, we’re now looking for the pathways that guide the cells outward growth and help them find each other.”
The mouse model provides a platform for evaluating how genetic changes, environmental exposures and pharmacological therapies, including drugs commonly used in the neonatal intensive care unit, affect lung development at the cellular level. It also is a discovery tool for new drugs that can promote tissue regeneration after injury.
All drugs have off-target, unintended effects. If, in the future, doctors could select treatments for their tiny patients with BPD that did not, as a side effect, interfere with alveologenesis, “or which would actually improve this process in the setting of injury, that would be fantastic,” Sucre said.
Traditionally, researchers have looked for “bad guy molecules” that cause disease, and “anti-bad guy molecules” to combat them. The problem is that the same molecule may be a villain in one situation and a hero in another.
While over-expression of the Wnt signaling pathway can lead to fibrosis (scar tissue formation) and cancer, for example, blocking the pathway entirely will prevent nerve growth.
Sucre said her lab is “keen to understand the cellular behaviors and dynamics in the more resilient mouse. What are the pathways in the resilient lung that can repair it after infection and injury? How do we bottle that?”
It is not a trivial matter to precisely cut and grow miniscule slices of lung tissue in the laboratory, Sucre said. The technique, which Sucre’s senior lab manager, Chris Jetter, perfected in neonatal mice, enabled visualization of fibroblast rings forming and contracting, and blood vessels growing into the developing alveoli.
Scientists from around the world have come to Nashville to train with Jetter. “He has established himself as a leading expert during the eight years that he has helped lead our research group,” Sucre said.
In the Vanderbilt Biophotonics Center, Bryan Millis, PhD, research associate professor of Biomedical Engineering and the paper’s co-senior author, led development of the custom-scanned oblique planar illumination scopes used in the study. This partnership“brings pre-commercial imaging to investigators tackling high impact questions,” Sucre said.
Philip Crooke, PhD, research professor of Mathematics, contributed to the quantitative analysis of the data for the paper, which was chosen as the cover article for the journal’s Feb. 24 issue.
Other co-authors currently at Vanderbilt and VUMC are Yeongseo Son, Erin Plosa, MD, John Benjamin, MD, Nicholas Mignemi, PhD, Meagan Ransom, MD, MPH, David Nichols, Susan Guttenberg, MD, Heather Pua, MD, PhD, Timothy Blackwell, MD, John Kozub, PhD, Anita Mahadevan-Jansen, PhD, Evan Krystofiak, PhD, Jonathan Kropski, MD, and Christopher Wright, DPhil.
The study was supported in part by National Institutes of Health grants T32 HL094296, K08 HL143051, R01 HL158556, K08 HL130595, R01 HL153246, R01 HL145372, P01 HL092470, R01 HL163195, R03 HL154287, K08 HL133484, and R01 HL157373, and by the Francis Family Foundation, the Chan Zuckerberg Initiative Imaging Scientists Program, and a Vanderbilt University Trans-Institutional Programs (TIPs) Award.
