PRMRP 2012 Investigator Vignette
Title: Lineage Analysis in Pulmonary Arterial Hypertension
Investigator: Peter Kao, MD, PhD; Stanford University
The title of the DoD grant was Lineage Analysis in Pulmonary Hypertension. In contrast to systemic hypertension, pulmonary hypertension is characterized by a cellular overgrowth in the center of the vessels.
The most important question to some, and the basis of the grant, is what is the nature and origin of the pathologic cell that is proliferating in the center of the blood vessels and obstructing the lumen of the blood vessels?
So we start with mice that carry fluorescent reporters. And so, these mice have been engineered so initially every cell expresses a red fluorescent protein tomato. And only those cells that are differentiated endothelial cells, they've switched the tomato red color to a green fluorescent color.
What we're seeing here is the inside of the capillaries in an alveolus of the mouse lung. Outlined here is a small pulmonary artery. The blue stain is a DAPI nuclear stain, and we also have a teal color. And the teal is an immuno-stain that detects smooth muscle actin.
What these panels are showing is that the green endothelial cells, a sub-set of them, around small pulmonary arteries actually express smooth muscle actin, so this is a new discovery. A sub-set of green endothelial cells around pulmonary arteries are expressing the teal smooth muscle actin.
During the injury process, subsequent to the development of experimental pulmonary hypertension, mouse gets a pneumonectomy, subsequently gets injected with monocrotaline pyrrole, six to ten weeks later it develops moderate pulmonary hypertension. When we look at the pathology of that mouse, we see that vessels are cross-sectionally filled in with green cells representing endothelial genetic lineage. When we stain with the antibodies of smooth muscle actin, we see that those luminal obliterating the pathologic cells in this disease are also expressing smooth muscle actin.
This is the power of the confocal microscopy. We can go through a physical section and take the time to determine the co-localization of the teal color, the smooth muscle actin on top of the GFP color, the genetic lineage marking in the endothelial cells. And you can see that the injured vessel really has some luminal narrowing and it's crawling, and compare that to the normal capillaries in the alveoli out here.
And I would hope to extend our studies from the animal model, and screen for therapies and drugs that would attenuate the inappropriate smooth muscle gene expression in the endothelial cells. So it's not quite anti-proliferation, and it's not quite vasodilation, but it's more a redirection back towards an endothelial phenotype.