Characterization of Blood Outgrowth Endothelial Cells (BOECs) from Hermansky-Pudlak Syndrome (HPS) Type 1
ISTH Academy. Purgatorio G. Jul 10, 2019; 274099; OC 77.4
Giulio Purgatorio
Giulio Purgatorio
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OC 77.4

Characterization of Blood Outgrowth Endothelial Cells (BOECs) from Hermansky-Pudlak Syndrome (HPS) Type 1

G. Purgatorio, E. Petito, E. Piselli, E. Falcinelli, G. Guglielmini, L. Bury, M. Malvestiti, P. Gresele
University of Perugia, Department of Medicine, Perugia, Italy

Main Topic: Immunothrombosis and Vascular Biology
Category: Stem Cells & Vascular Cell Growth

Background: HPS is an autosomal recessive disorder characterized by abnormal skin or ocular pigmentation and platelet dysfunction with altered biogenesis of lysosome-related organelles (LROs), including melanosomes and platelet dense granules. Nine genes (HPS1, AP3B1, and HPS3 to HPS9) have been identified as causative for HPS in humans. While several studies have explored the function of platelets, melanocytes, fibroblasts and their granules, no studies have explored endothelial cells from HPS.
Aims: To assess the impact of HPS1 mutation on endothelial cells.
Methods: BOECs were isolated from peripheral blood of a HPS1 patient and from healthy controls and characterized by immunofluorescence (IF) and flow-cytometry (FC) using antibodies against endothelial markers (VWF, CD31, CD146, VEGFR-2). Lysosomal-associated proteins LAMP1 and LAMP2 were assessed by IF and FC. Capillary-like structures (CLS)-sprouting was evaluted by angiogenesis-assay in Matrigel-coated plates. BOEC migration was assessed by the scratch assay on collagen-coated plates.
Results: Both HPS1 and control BOECs were positive for the endothelial markers CD31, VWF, CD146 and VEGFR-2 (CD309).
VWF and Weibel-Palade Bodies of HPS1 did not differ from controls, but BOECs from HPS1 showed higher surface expression of LAMP1 and LAMP2 compared to healthy controls (LAMP1: 26.4±2.08% vs 5.4±3.1%; LAMP2: 25.8±2.7% vs 7.4±3.3% p< 0.001), as assessed by both FC and IF.
Moreover, the scratch assay showed a significantly higher residual area in HPS1 (65.1±4.7% vs 33.1±4.3%; p< 0.05), showing defective EC migration. Finally, HPS1 BOECs formed a lower number of CLS (113±2.1 vs 153±2.7, p< 0.001) with shorter lenght (11.2±0.3mm vs 17±0.7mm, p< 0.001) and lesser branches (57.4±2.2 vs 75.2±2.9, p< 0.001) compared to controls, showing defective angiogenesis.
Conclusions: Our results show that HPS1 mutation alters the expression of lysosomal proteins and their trafficking in endothelial cells, resulting in an increased surface expression, but also impairs EC migration and neo-angiogenetic activity.

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