Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease

Wang, Jiong‐Wei; Bouwens, Eveline A.; Pintão, Maria Carolina; Voorberg, Jan; Safdar, Huma; Valentijn, Karine M.; Boer, Hetty C. de; Mertens, Koen; Reitsma, Pieter H.; Eikenboom, Jeroen

doi:10.1182/blood-2012-06-434373

Cited by 76 publications

(86 citation statements)

References 43 publications

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“…BOEC isolation culture. BOECs have been extensively used as a model for studying in vitro endothelial function in vascular disorders (29,30) and we have previously demonstrated their close functional and gene expression similarity to pulmonary artery endothelial cells (31). BOECs were isolated as previously described (44).…”

Section: Methodsmentioning

confidence: 99%

“…Blood outgrowth endothelial cells (BOECs) have been extensively used as a model for studying in vitro endothelial function in vascular disorders (29,30) with close functional and gene expression similarity to pulmonary artery endothelial cells (31). Previous work has shown enhanced expression and activity of Arg-2 in PAH patients (27,32).…”

Section: Changes In Acute Response To Hypoxia Caused By Loss Of Endotmentioning

confidence: 99%

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HIF2α–arginase axis is essential for the development of pulmonary hypertension

Cowburn

Crosby

Macías

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

156

158

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Hypoxic pulmonary vasoconstriction is correlated with pulmonary vascular remodeling. The hypoxia-inducible transcription factors (HIFs) HIF-1α and HIF-2α are known to contribute to the process of hypoxic pulmonary vascular remodeling; however, the specific role of pulmonary endothelial HIF expression in this process, and in the physiological process of vasoconstriction in response to hypoxia, remains unclear. Here we show that pulmonary endothelial HIF-2α is a critical regulator of hypoxia-induced pulmonary arterial hypertension. The rise in right ventricular systolic pressure (RVSP) normally observed following chronic hypoxic exposure was absent in mice with pulmonary endothelial HIF-2α deletion. The RVSP of mice lacking HIF-2α in pulmonary endothelium after exposure to hypoxia was not significantly different from normoxic WT mice and much lower than the RVSP values seen in WT littermate controls and mice with pulmonary endothelial deletion of HIF-1α exposed to hypoxia. Endothelial HIF-2α deletion also protected mice from hypoxia remodeling. Pulmonary endothelial deletion of arginase-1, a downstream target of HIF-2α, likewise attenuated many of the pathophysiological symptoms associated with hypoxic pulmonary hypertension. We propose a mechanism whereby chronic hypoxia enhances HIF-2α stability, which causes increased arginase expression and dysregulates normal vascular NO homeostasis. These data offer new insight into the role of pulmonary endothelial HIF-2α in regulating the pulmonary vascular response to hypoxia.A lveolar hypoxia affects vascular flow in the pulmonary vascular bed via an immediate vasoconstrictor response (hypoxic pulmonary vasoconstriction, or HPV) (1). This reduces perfusion of regions of the lung with lowered levels of airflow (2). In conditions including chronic obstructive pulmonary disease (3), idiopathic pulmonary fibrosis (4), and at high altitude (5), HPV probably contributes to persistent increases in pulmonary arterial pressures. This in turn is correlated with reduced plasticity of the vascular bed, sustained pulmonary vascular remodeling, and, ultimately, debilitating right ventricular hypertrophy (RVH) and failure (2).The hypoxia-inducible factors (HIFs) are transcription factors and key regulators of the molecular response to hypoxia. The targets of HIFs include genes controlling vascularization, cellular proliferation, migration, and metabolism (6-11). A wellcharacterized animal model of hypoxia-induced pulmonary hypertension involves exposure to chronic hypoxia (CH), typically 10-12% inspired oxygen. This results in extensive vascular remodeling, marked pulmonary hypertension and RVH over a period of a few weeks. Exposure to CH in rodents results in vasoconstriction and a pattern of vascular remodeling that is reminiscent of humans with hypoxia-associated pulmonary hypertension (12, 13).Mice that are hemizygous for either of the HIF isoforms, HIF-1α (14) or HIF-2α (15), have been shown to have attenuated pulmonary vascular remodeling following experimental CH. Conditional de...

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Section: Methodsmentioning

confidence: 99%

Section: Changes In Acute Response To Hypoxia Caused By Loss Of Endotmentioning

confidence: 99%

HIF2α–arginase axis is essential for the development of pulmonary hypertension

Cowburn

Crosby

Macías

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

156

158

View full text Add to dashboard Cite

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“…11 Prior to the availability of BOECs, endothelial cells could only be derived from explanted organs at time of death or organ transplantation, or isolated from the umbilical vein at birth. This reduced availability represented a serious limitation to understanding the biology of endothelial cells from patients with cardiovascular disorders, as well as the interactions between endothelial cells and either blood cells or mural cells.…”

Section: Introductionmentioning

confidence: 99%

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Ormiston

Toshner

Kiskin

et al. 2015

JoVE

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Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endotheliallike, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

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“…The impaired synthesis observed is supported by the increased FVIII/VWF ratio (1Á69) found in patients (Table I), which also suggests impaired synthesis of the VWF protein. Blood outgrowth endothelial cells (BOECs) from a patient carrying this variant also showed mainly short, round WPB and ER retention (Wang et al, 2013). The increased VWFpp/VWF:Ag ratio measured in patients with this p.Ser1285Pro mutation suggests that this VWF variant is also cleared faster .…”

Section: Discussionmentioning

confidence: 95%

“…The response to DDAVP was unfortunately not tested for patients with the p.Ser1285Pro mutation. However, one might speculate that patients will show a poor to partial response to DDAVP infusion based on our in vitro data and data obtained from BOECs of a VWD patient (Wang et al, 2013). We anticipate that the regulated secretion in patients carrying this mutation might be quantitatively impaired due to retention of VWF in the ER.…”

Section: Discussionmentioning

confidence: 99%

Storage and secretion of naturally occurring von Willebrand factor A domain variants

et al. 2014

Self Cite

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SummaryVon Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.

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Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease

Abstract: Key Points Isolation of BOECs from multiple patients with VWD is feasible, and the study of BOECs helps explain the pathogenic complexity of VWD. Abnormalities in WPB biogenesis and exocytosis and defects in VWF string formation correlate with the phenotypic features of patients with VWD.

Cited by 76 publications

References 43 publications

HIF2α–arginase axis is essential for the development of pulmonary hypertension

HIF2α–arginase axis is essential for the development of pulmonary hypertension

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Storage and secretion of naturally occurring von Willebrand factor A domain variants

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