Circulating Versus Progenitor Endothelial Cells Levels Are Abnormal in Patients with Different Types of Von Willebrand Disease and Correlate with Markers of Angiogenesis
Authors: Gritti Giuseppe , Federici Augusto B , Cortelezzi Agostino , Baronciani Luciano , Lonati Silvia , Punzo Margherita , Silvestris Ilaria , Garavaglia Rosanna , Lambertenghi Giorgio , Mannucci Pier M 
Published: April 2020

Abstract
Background:Von Willebrand disease (VWD) is the most common inherited bleeding disorder that is characterized by deficiency or abnormality of von Willebrand factor (VWF). VWF, synthesized by endothelium and magakaryocytes, circulates in plasma and is present in sub-endothelium and platelets: therefore it is an ideal marker for endothelial formation/damage and megakaryocytopoiesis. Circulating (CEC) and progenitor (EPC) endothelial cells have been also proposed as markers of bone marrow-derived angiogenesis.
Aims of the study: to evaluate the association of CEC/EPC with cellular and circulating VWF, we have measured the number of CEC/EPC together with VWF and cytokines involved in angiogenesis in a cohort of 84 patients with different VWD types.
Methods: Eighty-four VWD patients were diagnosed according to the recommendations of the Scientific Standardization Committee Sub-committee on VWF of the ISTH. The VWD cohort was composed by the following VWD types (number): 1 (27); 2A (10); 2B (23); 2M (17), 3 (7). Twenty healthy individuals were used as controls. CEC (CD146+, CD31+,CD45−) and EPC (CD34+, CD133+, CD45−) were evaluated by cytometry. Circulating levels of VEGF, E-selectin and EPO were determined by ELISA. Both CEC/EPC and cytokines were tested in blind. i.e. without knowing in advance the type of VWD diagnosis.
Results: Median (range)[* = p &lt;0.05] of CEC/EPC measured in controls and in different VWD types are shown below together with those of VEGF, E-selectin and EPO (Table).
Patients (n) CECs (cells ×106/l) EPCs (cells ×106/l) VEGF (pg/ml) E-Selectin (ng/ml) EPO (mIU/ml) Controls (20) 13.4 (4.3–33.0) 0.24 (0.04–0.60) 224 (92–392) 28 (14.6–34.2) 7.1 (3,0–16,6) All VWD (84) 20.0 (2.0–133.2)* 0.09 (0.00–1.14)* 334 (88–1286)* 34.1 (13.0–125.5)* 10.5 (1.7–53.0)* VWD1 (27) 19.2 (4.2–133.2) 0.08 (0.00–1.14)* 296 (121–1103)* 28.7 (13.0–74.3) 11.9 (1.7–26.6)* VWD2A/2M (27) 16.5 (2.0–58.9)* 0.08 (0.00–0.53)* 371 (88–1286)* 41.9 (19.6–117.3)* 10.2 (6.3–44.7)* VWD2B (23) 30.1 (9.0–108.8)* 0.13 (0.00–0.96)* 330 (142–675)* 33.8 (13.8–103.9)* 12.9 (2.9–53.0)* VWD3 (7) 41.3 (5.2–78.9)* 0.13 (0.00–0.37) 475 (131–676) 33.8 (22.6–125.5)* 10.5 (7.2–19.0)
The global VWD population had higher CEC (p=.002) and lower EPC (p=.001) than controls. These data were associated with high VEGF (p=.003), E-selectin (p=.001) and EPO (p=.001) levels. As far as VWD types, VWD1 patients showed slightly high CEC (p=.06), normal E-selectin, markedly low EPC (p=.002) and high VEGF (p=.02). VWD2A/2M, grouped together, showed higher CEC (p=.05) and low EPC (p=.009) together with high VEGF (p=.005), EPO (p=.004) and markedly higher E-selectin (p&lt;.0001). Conversely, VWD2B, characterized by enhanced interactions with platelet glycoproteins Ib (GpIb), and VWD3, by virtually complete deficiency of VWF, showed high CEC (p=.0002 and .01) and relatively reduced EPC (p=.03 and .05) with elevated VEGF (p=.01 and .13), E-selectin (p=.002 and .02) and EPO (p=.005 and 0.1).
Discussion and conclusions: Based on these results, we can conclude that CEC are increased in VWD patients, especially in VWD2B and 3: high CEC are associated with increased levels of cytokines involved in angiogenesis (up-regulation). Conversely, EPC are always decreased in VWD patients, especially in VWD1 and VWD2A/2M, suggesting down-regulation of bone marrow-derived angiogenesis. These preliminary data might be important to better understand the role of VWF in the pathogenesis of different VWD types as well as in bone marrow-derived angiogenesis