Identifying a proliferating repairman for tissue in damaged lungs

Veins in the lungs, or pulmonary veins, play a critical role not only in lung functioning but also in maintaining sufficient oxygen in tissue throughout the body. When a person sustains pulmonary injury from an illness like influenza or COVID, repair of blood vessels and the creation of new ones is vital to meet oxygen demands; however, research in these areas remains underexplored.

Researchers from the University of Pennsylvania’s School of Veterinary Medicine and Perelman School of Medicine, Children’s Hospital of Philadelphia (CHOP), and Vanderbilt University Medical Center have been studying the role of pulmonary venous endothelial cells (VECs) in endothelial regeneration after adult lung injury. VECs line the inside of blood vessels in the lungs, playing an essential role in blood flow and angiogenesis, the creation of new blood vessels.

Their new paper shows that venous endothelial cells can help fix damaged blood vessels in the lungs. The researchers found that following influenza, COVID, and hyperoxia injury, VECs proliferate into the adjacent capillary bed—a network of blood vessels facilitating gas exchange—and contribute to its regeneration.

They also show that VECs differentiate into capillary cells, and that this remodeling is a response to lung injury, not one that occurs during normal lung development after birth. Their findings are published in Nature Cardiovascular Research.

“A lot of patients who encounter respiratory viruses, especially if they’re immunocompromised, can develop something called acute respiratory distress and end up in the intensive care unit,” says first author Joanna Wong, a doctoral student in the lab of Andrew E. Vaughan at Penn Vet. “Trying to figure out ways to promote the regeneration of their vascular bed or lungs in general would advance modern medicine and patient care.”

Vaughan is co-senior author on the study with David B. Frank, a pediatric cardiologist and assistant professor at CHOP. Vaughan notes that most of the people with COVID who died in the ICU died from acute respiratory distress syndrome, which has a mortality rate above 30%. “Now that we have identified an important progenitor population, we might be able to figure out how to mess around with the cells in those veins and improve their ability to contribute to repair,” Vaughan says, making these cells a potential target for therapeutics.

The inspiration for this study came from research in zebrafish and mice. Vaughan explains that past studies in these animals suggested that “at least in some organs in some contexts, a lot of the capillary bed is built by expansion of the veins.” But nobody had ever looked at the lungs.

Wong also points to work done in the Vaughan Lab, before she joined. Then, postdoctoral fellow Gan Zhao led a study that found knocking out a certain vein-specifying transcription factor exacerbated lung injury and reduced proliferation of endothelial cells.

Taken together, Vaughan and Wong say, these studies led them and their colleagues to hypothesize that endothelial cells lining the veins in the lungs would also contribute to repair of the capillary bed after injury.

Veins branch out into the capillary bed after lung injury from influenza. (Image: Courtesy of Joanna Wong)

There was one problem: There were no existing tools to single out these cells and follow them over time. Wong set about going through single-cell RNA sequencing data of pulmonary endothelial cells at zero, 20, and 30 days after influenza injury, information the lab had previously generated. She identified the perfect marker: a gene called Slc6a2 that only showed up in pulmonary veins. They teamed up with Frank to proceed with generating a mouse model based on this gene.

“It was really weird, because it’s also a norepinephrine transporter, which is normally associated with neurons, and we’re still not sure why it would be expressed in the pulmonary veins,” she says. But it was fortuitous, and she was able to use Slc6a2 to track the fate of VECs in a genetically modified mouse model.

Looking ahead, Wong says researchers are trying to determine what mechanisms contribute to VEC sprouting and are also looking at angiogenesis in other contexts, such as cancer.

Vaughan says the methodology from this paper doesn’t work until after birth, but he would like to know if, during lung development, early embryonic veins play a role in building the rest of the blood vessels.

Joanna Wong is a developmental, stem cell, and regenerative biology doctoral candidate in the Cell & Molecular Biology Graduate Group at the University of Pennsylvania.

Andrew E. Vaughan is an associate professor of biomedical sciences in the University of Pennsylvania School of Veterinary Medicine and assistant professor in the Institute for Regenerative Medicine at the Perelman School of Medicine.

David B. Frank is an assistant professor of Pediatrics in the Department of Pediatrics at Penn Medicine and an attending physician in the Children’s Hospital of Philadelphia.

The other co-authors are Stephanie Adams-Tzivelekidis, Maria E. Gentile, Nicolas P. Holcomb, Sara Kass-Gergi, Xinyuan Li, Meryl Mendoza, Madeline Singh, and Gan Zhao of Penn Vet and Penn Medicine; Hongbo Wen, Prashant Chandrasekaran, and Sylvia N. Michki of Children’s Hospital of Philadelphia; Alan T. Tang of Penn Medicine; and Nicholas M. Negretti and Jennifer M.S. Sucre of Vanderbilt University Medical Center.

This research was supported by the National Heart, Lung, and Blood Institute (grants R01HL164350 and R56HL167937); the Ayla Gunner Prushansky Fund; and a National Service Research Award Individual Predoctoral Fellowship.