3D visualization of vascular lesions and the role of versican in pulmonary arterial hypertension

Abstract: Pulmonary arterial hypertension (PAH) is a condition that leads to lethal right heart failure. Available treatments provide relief but are far from curative and the vascular remodeling in PAH has been difficult to decipher. Although pathognomonic to PAH, the plexiform lesions are difficult to define, and there is no consensus regarding their potential hemodynamic impact. Versican is an extracellular matrix proteoglycan known to accumulate in systemic vascular disease, often colocalized with hyaluronan. Previous results have shown that versican is abundant in vascular lesions of PAH. However, its functional role warrants investigation.This PhD project have had two separate aims, of equal importance. A novel imaging technique called synchrotron- based phase-contrast micro-CT (SPμCT) was evaluated for its potential use as a tool for virtual histology of pulmonary vascular pathology. SPμCT was used in all five projects. Versican was studied in human PAH tissue by immunofluorescence and in situ hybridization, and its function in disease development was evaluated in murine models of pulmonary hypertension.SPμCT imaging revealed the possibility of non-destructive 3D imaging of paraffin-embedded pulmonary tissue with clear contrast and high resolution. With this technique, we could demonstrate the presence of patent intrapulmonary bronchopulmonary anastomoses in a subgroup of pediatric PAH called alveolar capillary dysplasia and confirm the identity of misaligned pulmonary veins as dilated bronchial veins. SPμCT imaging of adult idiopathic PAH (IPAH) tissue enabled 3D visualization of plexiform lesions. Significant plexiform lesion heterogeneity was identified, as well as proof of shunting between the pulmonary and bronchial circulation within some lesions. These results led to a novel classification of plexiform lesions. The same technique was used for a rat model of PAH (Sugen5416/hypoxia) where for the first time an animal model has been shown to have the full spectrum of human type plexiform lesions, including shunt-type lesions.GAG AR-a and -b containing isoforms of versican were found to be produced and deposited in lesions of PAH. Hyaluronan was found abundant in the neointima of vascular lesions, consistently colocalizing with versican. Known neoepitopes of versican, generated by enzymatic cleavage, were observed and the staining pattern indicated further enzymatic cleavage of the core protein. Serum samples from IPAH and control patients revealed significantly elevated levels of versican G3 in patient blood, making it a possible biomarker. Neither a global partial (two isoforms) or inducible complete (all isoforms) knockdown of versican affected pulmonary hypertension in mice exposed to chronic hypoxia, perhaps due to the lack of occlusive lesion formation in this model. SPμCT imaging revealed gradual formation of occlusive vascular lesions, with versican deposition, in a mouse model where exposure to house dust mite generates pulmonary hypertension. These results indicate that this model may be more suitable for studies of the role of versican in vivo.In conclusion, SPμCT imaging and the novel classification of plexiform lesions have the potential to advance the PAH field significantly, and versican G3 is a potential biomarker for pulmonary vascular remodeling.