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A2076 - The Role of αGAG-Containing Versican Isoforms in Pulmonary Hypertension
Author Block: C. Norvik1, C. Westoo1, M. Orcholski2, Y. Chang3, M. T. Dours-Zimmermann4, S. S. Apte5, V. De Jesus Perez2, K. Tran-Lundmark1; 1Department of Experimental Medical Science, Lund University, Lund, Sweden, 2Division of Pulmonary and Critical Care Medicine, Stanford Univ, Stanford, CA, United States, 3Department of Pediatrics, Chang Gung Memorial Hospital, Taipei, Taiwan, 4Department of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland, 5Department of Biomedical Engineering, Lerner Research Institute, Cleveland, OH, United States.
Rationale: Pulmonary hypertension leads to right ventricular failure and death if left untreated. Available treatment has increased survival, but the long-term prognosis is still poor and treatment which targets pulmonary vascular remodeling more specifically is needed. Proteoglycans are proteins with attached glycosaminoglycan (GAG) chains which, among other functions, can regulate the bioavailability of growth factors and cytokines. We have recently demonstrated accumulation of the proteoglycan versican in human pulmonary arterial hypertension (PAH) and analysis by whole exome sequencing showed that VCAN is one of the most frequently mutated genes in PAH. The versican core protein contains a G1 domain, a G3 domain, and GAG-binding regions, named αGAG and βGAG domains. Alternative splicing generates at least four isoforms which vary in the GAG-attachment regions. Here, we investigate the role of αGAG-containing isoforms (V0 and V2) in pulmonary hypertension.
Methods: The point mutations found by whole exome sequencing were analyzed in detail. Immunohistochemistry for different domains of versican was performed on PAH tissue. Pulmonary hypertension was induced in versican V0/V2 knockouts, a mouse strain lacking the two αGAG-containing isoforms. 36 mice were divided into four groups; WT and mutant normoxia and hypoxia. Hypoxic groups were kept in 10% O2 for 4 weeks. Right ventricular systolic pressure (RVSP) was measured. Hearts and lungs were collected for estimation of right ventricular hypertrophy using RV/(LV+S) and for quantification of pulmonary vascular muscularization.
Results: All, but one, of the point mutations in the VCAN gene were in the αGAG-encoding region. None of the mutations were in places predicted to affect versican cleavage by ADAMTS enzymes. Both αGAG and βGAG domains were shown to be present in vascular lesions of PAH patients, with slightly different patterns of distribution, confirming the presence of αGAG-containing isoforms in human PAH. Following hypoxia both V0/V2 knockouts and WT showed significant elevations of RVSP and increases in RV/(LV+S) ratio, with no significant differences between the genotypes. Analysis of vascular muscularization is ongoing.
Conclusion: Versican αGAG point mutations are common in PAH, and αGAG-containing isoforms can be detected in vascular lesions. However, lack of versican isoforms V0 and V2 does not affect the development of murine hypoxia-induced pulmonary hypertension, possibly due to species differences. However, it is also possible that the point mutations have secondary effects leading to an increase in V1. We plan to explore this further by studying pulmonary hypertension in inducible complete versican knockouts.