Weibel Palade Research Articles

Abstract 1063: G Protein 12 Alpha Subunit Covalent Binding To Alpha-snap And Its Potential Role In Von Willebrand Factor Secretion

Rationale: α-SNAP is part of a large 20S complex composed of 3 SNARE, 4 α-SNAP, and 6 N-ethylmaleimide sensitive factor proteins that together regulate endothelial cell Weibel Palade body fusion and exocytosis of von Willebrand factor (vWF). We previously showed that Gα12 is essential for basal and thrombin-induced vWF secretion, that GST-α-SNAP pulldown of Gα12 is dependent on N-terminal domain aa 10-15 in Gα12, and that a myristoylated 6 aa α-SNAP Binding Domain Gα12 peptide (Myr-SBD6) reduces vWF secretion, thrombo-inflammation, and lethality in septic mice. Here, using direct binding assays (Surface Plasmon Resonance (SPR), AlphaLISA, and mass spectrometry (MS)), we assessed the molecular underpinnings of SBD6 and modified higher affinity peptide (SBD15) binding directly to α-SNAP. Methods: GST-α-SNAP was incubated alone or with SBD6, SBD15-PEG-biotin, or SBD6 with C to S mutation and then evaluated by Maldi MS. SPR was performed for both SBD6 and SBD15 to assess binding affinity ( K D ) and kinetics ( k a and k d ). After demonstrating the covalent linkage of α-SNAP with SBD15-PEG-biotin, cysteine mapping was performed to identify the SBD binding site on α-SNAP. Free cysteines in SBD15-biotin conjugated α-SNAP were capped with N-ethylmaleimide, disulfide bonds were reduced with DTT, reduced Cys residues were capped with iodoacetamide (IAA), and tryptic peptide digests were analyzed by LC/MS/MS. Results: Maldi MS studies showed that SBD15-PEG-biotin exhibited covalent binding to GST-α-SNAP. Further, proteomic analysis of Cys mapped fragments identified a peptide containing C103, KADPQEAINC 103 LMR, that was modified by NEM in control samples and shifted in the presence of SBD15, indicating modification by IAA in α-SNAP - SBD15 conjugates. Further, the C103S α-SNAP mutant was unable to bind to SBD6, and C11S-SBD15-PEG-biotin was unable to bind to α-SNAP. Conclusion: We propose that amino-terminal Gα12 Cys11 covalently binds to α-SNAP Cys103 and that this plays a critical role in the mechanism regulating vWF secretion. The discovery of this disulfide binding mechanism between Gα12 and α-SNAP opens up new avenues for therapeutic intervention in thrombotic disorders associated with elevated plasma vWF.

The proteasome inhibitor carfilzomib exerts anti-inflammatory and antithrombotic effects on the endothelium

BackgroundCarfilzomib (CFZ) is a second-generation proteasome inhibitor used to treat multiple myeloma. Potent inhibition of the proteasome results in chronic proteotoxic endoplasmic reticulum (ER) stress, leading to apoptosis. While CFZ has improved survival rates in multiple myeloma, it is associated with an increased risk of cardiovascular adverse effects. While this has been putatively linked to cardiotoxicity, CFZ could potentially also exhibit adverse effects on the endothelium. ObjectivesTo investigate the effects of CFZ on the endothelium. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with CFZ, and expression of relevant markers of ER stress, inflammation, and thrombosis was measured and functionally assessed. ResultsCFZ failed to induce ER stress in HUVECs but induced the expression of Kruppel-like factor 4, endothelial nitric oxide synthase, tissue plasminogen activator, and thrombomodulin and reduced tumor necrosis factor alpha (TNFα)-mediated intercellular adhesion molecule 1 and tissue factor expression, suggesting a potential protective effect on the endothelium. Consistent with these observations, CFZ reduced leukocyte adhesion under shear stress and reduced factor Xa generation and fibrin clot formation on the endothelium following TNFα treatment and inhibited von Willebrand factor (VWF) and angiopoietin-2 exocytosis from Weibel–Palade bodies. Subsequently, CFZ inhibited the formation of VWF-platelet strings, and moreover, media derived from myeloma cell lines induced VWF release, a process also inhibited by CFZ. ConclusionThese data demonstrate that CFZ is unable to induce ER stress in confluent resting endothelial cells and can conversely attenuate the prothrombotic effects of TNFα on the endothelium. This study suggests that CFZ does not negatively alter HUVECs, and proteasome inhibition of the endothelium may offer a potential way to prevent thrombosis.

Transcriptional and functional profiling identifies inflammation and endothelial-to-mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of endothelial colony forming cells

BackgroundEndothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease. ObjectivesTherefore, we aimed to determine phenotypic and gene expression differences between control ECFCs. MethodsA total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed. ResultsThrough hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel–Palade bodies. Migration speed in cluster 1 was also significantly higher. ConclusionWe observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel–Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders.

Approaches to induce the maturation process of human induced pluripotent stem cell derived-endothelial cells to generate a robust model.

Endothelial cells generated from induced pluripotent stem cells (hiPSC-ECs) show the majority of endothelial cell characteristics and markers, such as cobblestone morphology and the expression of VEGF and VE-cadherin. However, these cells are failing to show a mature endothelial cell phenotype, which is represented by the low expression and production of von Willebrand Factor (VWF) leading to the round morphology of the Weibel Palade Bodies (WPBs). The aim of this study was to improve the maturation process of hiPSC-ECs and to increase the levels of VWF. hiPSC-ECs were differentiated by a standard differentiation protocol from hiPSCs generated from healthy control donors. To induce maturation, the main focus was to increase the expression and/or production of VWF by the adjustment of potential parameters influencing differentiation and maturation. We also compared alternative differentiation protocols. Cells were analyzed for the expression of endothelial cell markers, WPB structure, and the production and secretion of VWF by flow cytometry, confocal microscopy and ELISA. The generated hiPSC-ECs have typical endothelial cell surface expression profiles, with low expression levels of non-endothelial markers as expected. Co-culture with pericytes, varying concentrations and timing of differentiation factors, applying some level of flow, and the addition of HDAC inhibitors did not substantially improve maturation of hiPSC-ECs. Transfection with the transcription factor ETV2 to induce a faster hiPSC-EC differentiation process resulted in a limited increase in VWF production, secretion, and elongation of WPB structure. Alternative differentiation protocols had limited effect. hiPSCs-ECs have the potential to show a more mature endothelial phenotype with elongated WPBs after >30 days in culture. However, this comes with limitations as there are very few cells detected, and cells are deteriorating after being in culture for extended periods of time.

Coronary microthrombi in the failing human heart: the role of von Willebrand factor and PECAM-1

The recognition of microthrombi in the heart microcirculation has recently emerged from studies in COVID-19 decedents. The present study investigated the ultrastructure of coronary microthrombi in heart failure (HF) due to cardiomyopathies that are unrelated to COVID-19 infection. In addition, we have investigated the role of von Willebrand factor (VWF) and PECAM-1 in microthrombus formation. We used electron microscopy to investigate the occurrence of microthrombi in patients with HF due to dilated (DCM, n = 7), inflammatory (MYO, n = 6) and ischemic (ICM, n = 7) cardiomyopathy and 4 control patients. VWF and PECAM-1 was studied by quantitative immunohistochemistry and Western blot. In comparison to control, the number of microthrombi was increased 7–9 times in HF. This was associated with a 3.5-fold increase in the number of Weibel–Palade bodies (WPb) in DCM and MYO compared to control. A fivefold increase in WPb in ICM was significantly different from control, DCM and MYO. In Western blot, VWF was increased twofold in DCM and MYO, and more than threefold in ICM. The difference between ICM and DCM and MYO was statistically significant. These results were confirmed by quantitative immunohistochemistry. Compared to control, PECAM-1 was by approximatively threefold increased in all groups of patients. This is the first study to demonstrate the occurrence of microthrombi in the failing human heart. The occurrence of microthrombi is associated with increased expression of VWF and the number of WPb, being more pronounced in ICM. These changes are likely not compensated by increases in PECAM-1 expression.

Evoked Weibel-Palade Body Exocytosis Modifies the Endothelial Cell Surface by Releasing a Substrate-Selective Phosphodiesterase.

Weibel Palade bodies (WPB) are lysosome-related secretory organelles of endothelial cells. Commonly known for their main cargo, the platelet and leukocyte receptors von-Willebrand factor (VWF) and P-selectin, WPB play a crucial role in hemostasis and inflammation. Here, the authors identify the glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) as a WPB cargo protein and show that GDPD5 is transported to WPB following uptake from the plasma membrane via an unique endocytic transport route. GDPD5 cleaves GPI-anchored, plasma membrane-resident proteins within their GPI-motif, thereby regulating their local activity. The authors identify a novel target of GDPD5 , the complement regulator CD59, and show that it is released from the endothelial surface by GDPD5 following WPB exocytosis. This results in increased deposition of complement components and can enhance local inflammatory and thrombogenic responses. Thus, stimulus-induced WPB exocytosis can modify the endothelial cell surface by GDPD5-mediated selective release of a subset of GPI-anchored proteins.

Targeted Inhibition of Phosphodiesterase (PDE) 4 As a Novel Therapy to Increase Endothelial Cell cAMP and Trigger Weibel Palade Body Exocytosis

Background Desmopressin (DDAVP) is widely used in the treatment of VWD and other bleeding disorders. DDAVP activates V2 receptors (V2R) on endothelial cells (EC) to stimulate cAMP generation and Weibel-Palade body (WPB) exocytosis. However, DDAVP has inherent limitations, including lack of oral formulation, side-effects related to renal V2R agonism and sub-optimal responses in some patients. Aims To identify previously approved drugs with capacity to trigger cAMP-dependent WPB secretion in EC to repurpose as novel hemostatic therapeutic agents. Methods Candidate agents were identified by mechanistic screening of FDA and EMA-approved drug databases and rationalized for translational relevance. For each class, prototypical drugs were tested in vitro for capacity to induce VWF release from macrovascular (HUVEC) and microvascular (HMVEC-L) EC (VWF antigen [Ag], propeptide [pp] and collagen binding [CB] in supernatant at 1 hour, expressed as fold increase relative to untreated control [NC]). VWF string formation on the surface of treated EC under flow conditions was determined using fluorescent anti-VWF antibodies and confocal microscopy. Finally, the effects of candidates on platelet aggregation were determined by light transmission aggregometry (LTA). Results and Discussion Mechanistic screening and preliminary in vitro evaluation determined a lead candidate drug class: PDE-4 inhibitors. Previous studies have shown that cAMP-hydrolyzing PDE isoforms 2, 3 and 4 are expressed in EC and that non-selective PDE inhibition (PDE-I) with IBMX alone can elevate cAMP in EC sufficiently to induce VWF release. Consistently, we observed that treatment of EC with IBMX significantly enhanced VWF secretion (median fold increase VWF:Ag vs NC:1.49, p<0.0001, Fig.1). However, PDE isoforms 2 and 3 are also present in platelets, where increased cAMP activity inhibits platelet aggregation. We confirmed by LTA that IBMX significantly attenuated TRAP-6-induced platelet aggregation, limiting the therapeutic relevance of non-selective PDE-I as VWF-raising agents. We next evaluated the capacity of isoform-selective PDE-I to induce VWF release. Selective inhibition of PDE-2 (with EHNA) and PDE-3 (with Cilostamide) had no significant effect (p>0.999, Fig.1). In contrast, selective PDE-4 inhibition with Roflumilast (ROF) resulted in a dose-dependent increase in VWF:Ag secretion in both HUVEC (1.51 fold, p<0.0001, Fig.1) and HMVEC-L (1.75 fold, p=0.007). Notably, these effects were also seen with ROF concentrations in the nanomolar range, consistent with plasma levels following single oral dose in healthy subjects. In keeping with a role for PDE-4 in regulating cAMP-mediated WPB exocytosis, ROF treatment of EC resulted in a proportionate increase in VWFpp secretion (1.74 fold, p=0.0002). Furthermore, pre-incubation of HUVEC with ROF triggered VWF string formation under flow conditions, with strings visualized in most fields of view (Fig.2, 80% ROF vs 0% NC, p=0.0003). Consistently, following treatment with ROF, increased VWF:Ag was accompanied by a parallel rise in VWF:CB in the supernatant (1.50 fold, p=0.0061), confirming the secretion of hemostatically active VWF multimers. Critically, however, ROF had no inhibitory effect on platelet aggregation, consistent with lack of PDE-4 isoform activity in platelets. Finally, ROF combined with either histamine (HIS) or thrombin (THR) had synergistic effects on VWF release, suggesting ROF could prime EC to physiological agonists at times of hemostatic challenge (3.4 fold HIS vs 5.17 fold HIS+ROF, p<0.0001; 3.7 fold THR vs 4.3 fold THR+ROF, p=0.0060). In addition, ROF exhibited synergistic activity with the cAMP-raising agent Isoprenaline compared to ROF alone (1.77 vs 1.40 fold, p=0.0040, Fig.1), suggesting combination therapy as a novel therapeutic approach. Conclusion Drug repurposing may allow for rapid and cost-effective translation into clinical practice. Importantly, PDE-4 inhibitors such as ROF are currently licensed oral agents for treating Psoriasis and COPD. Our novel findings identify PDE-4 as a critical endothelial PDE isoform that regulates WPB exocytosis and VWF secretion. Moreover, we demonstrate that ROF stimulates VWF release in vitro without deleterious effects on platelet aggregation. Our data highlight the therapeutic potential of PDE-4 inhibitors, either alone or in combination with DDAVP, in the treatment of VWD.

Ralb and Exocyst Regulate Endothelial Cell Von Willebrand Factor Release

Background: Identification of novel therapeutic targets for pharmacologic modulation of circulating von Willebrand factor (vWF) would be invaluable in treatment of von Willebrand disease, but also in other pathophysiologic processes where vWF plays a vital role. These include cardiovascular disease, cancer metastasis, angiogenesis, and smooth muscle proliferation. vWF is almost exclusively produced by the endothelial cells and stored in specialized secretory granules, Weibel Palade bodies (WPBs). We have previously shown that loss of the exocyst complex, an essential multi-subunit trafficking protein, which physically tethers secretory granules to the plasma membrane in yeast, increases vWF release. Small GTPases act as regulatable molecular switches. Cycling between GTP-bound ('on') and GDP-bound ('off') states, they control all essential cellular processes. Among these, Ral GTPase has previously been shown to be important in WPB secretion and vWF release. Since Ral specifically regulates exocyst in other cellular functions, we hypothesized that exocyst is also under direct control of Ral in vWF release. Methods: All experiments were performed on human umbilical vein endothelial cells (HUVECs). RalA/B and exocyst depletion were performed via targeted siRNAs. vWF antigen was estimated using ELISA. RalB activation was determined by a commercial pulldown assay specific for the activated form of RalB. Expression of FLAG-tagged RalB was performed by lentiviral transduction. RalB mutants were generated using site-directed mutagenesis of FLAG-RalB (WT). Spinning disc confocal microscope was used for immunofluorescence (IF). Immuno-electron microscopy was performed using immune-gold labeling. FLAG-pulldown and immunoprecipitation (IP) was performed on cell lysates using antibody-labeled magnetic beads. Results: RalB but not RalA depletion, or degradation with the RalB-selective inhibitor dihydroartemisinin, significantly impaired thrombin-induced vWF exocytosis (55±7% decrease). RalB was activated (GTP-bound) and phosphorylated upon thrombin stimulation of HUVECs. Correspondingly, expression of constitutively active RalB-G23V mutant, which stays in a GTP-bound state, significantly increased vWF exocytosis compared to RalB-WT (69±4.6% increase). IF and EM confirmed co-localization of RalB and exocyst on WPBs. Unexpectedly, FLAG pulldown and protein identification by mass spectrometry demonstrated that RalB-WT (GDP-bound;“off”) but not RalB-G23V (GTP-bound;“on”) interacts with exocyst, confirmed by immunoblotting. IP of resting and thrombin-stimulated native HUVEC lysates with RalB antibodies, as well as reverse-IP with exocyst antibodies confirmed this differential binding. Exocyst binding site mutation (RalB-WTD49E) and transduction of these mutants in HUVECs resulted in significantly augmented vWF exocytosis (40±3% increase), like constitutively active RalB-G23V mutant. Moreover, IF showed uniform cytoplasmic distribution of WPB cigars in RalB-WT HUVECs, whereas in RalB-WTD49E mutants WPBs were found at the plasma membrane, like RalB-G23V. This confirms that RalB-free exocyst tethers WPB to the plasma membrane ( Figure 1). Serine phosphorylation site mutation (RalB-WTS198A), previously shown to regulate GTPase activity of RalB, hence GTP-GDP cycling, also significantly decreased RalB exocyst binding, in turn increasing vWF release. This shows that post-translational phosphorylation of RalB at serine 192, which is also accelerated by thrombin activation of endothelial cells, regulates RalB-exocyst interaction, hence vWF release. Conclusions: We have identified an important regulatory role of RalB in exocyst-dependent WPB secretion and vWF release. WPB localized exocyst is associated with RalB in resting endothelium. Endothelial cell activation switches RalB ‘on’, and exocyst, now uncoupled from GTP-bound RalB, is free to tether WPBs to the plasma membrane and facilitate vWF release.

Von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel–Palade bodies

BackgroundThe von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel–Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. MethodsStatic-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. ResultsStatic-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app ∼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF–Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. ConclusionsTogether, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction.

Clinical significance of assessing ADAMTS-13 and von Willebrand factor level in COVID-19 convalescent pregnant women

Introduction. Coronavirus infection is associated with severe endotheliopathy, thromboinflammation and immunothrombosis leading to excessive release of von Willebrand factor (vWF) multimers from Weibel–Palade bodies, which can affect activity of ADAMTS-13 metalloproteinase (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) and the ADAMTS-13/vWF axis previously shown by us to be altered in non-pregnant women with severe COVID-19. Aim: to study a clinical role of hemostasis activation particularly ADAMTS-13/vWF axis in pregnant women after COVID-19. Materials and Methods. A prospective case–control study was conducted with pregnant women (n = 135) divided into 3 groups: group 1 included 45 women with prior COVID-19 during pregnancy, group 2 – 45 women in the acute phase of the infection during pregnancy, group 3 – 45 healthy pregnant women. The level of vWF and ADAMTS-13 was assessed in all patients. Results. The concentration of vWF antigen (vWF:Ag) in the acute period of the disease in pregnant women with COVID-19 was significantly higher compared to the control group (p < 0.001). ADAMTS-13 level in pregnant women after COVID-19 did not differ from that of in control group, while vWF level was significantly higher in 66.7 % (30/45). The ADAMTS-13/vWF ratio was increased and significantly differed both in pregnant patients during the acute period of the disease (p < 0.001) and pregnant women after infection (p = 0.0002) compared with the control group. Conclusion. Our results show that endotheliopathy was prominently manifested in pregnant women with COVID-19 and persisted for several months after disease. The ADAMTS-13/vWF ratio determines the pathway functioning, the risk of microcirculation disorders and clinical complications.

Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts.

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMECD), or from nominally healthy donors (controls; HCMECC). Using fluorescence microscopy, WPBs in HCMECC (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMECD (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMECD revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMECD WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMECD were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

Is Endothelial Activation a Critical Event in Thrombotic Thrombocytopenic Purpura?

Thrombotic thrombocytopenic purpura (TTP) is a severe thrombotic microangiopathy. The current pathophysiologic paradigm suggests that the ADAMTS13 deficiency leads to Ultra Large-Von Willebrand Factor multimers accumulation with generation of disseminated microthrombi. Nevertheless, the role of endothelial cells in this pathology remains an issue. In this review, we discuss the various clinical, in vitro and in vivo experimental data that support the important role of the endothelium in this pathology, suggesting that ADAMTS13 deficiency may be a necessary but not sufficient condition to induce TTP. The "second hit" model suggests that in TTP, in addition to ADAMTS13 deficiency, endogenous or exogenous factors induce endothelial activation affecting mainly microvascular cells. This leads to Weibel-Palade bodies degranulation, resulting in UL-VWF accumulation in microcirculation. This endothelial activation seems to be worsened by various amplification loops, such as the complement system, nucleosomes and free heme.

Quantitative analysis of Weibel-Palade bodies.

Weibel Palade bodies (WPBs) are vesicles found in endothelial cells which carry the multimeric protein von Willebrand factor (VWF). As cellular confluency has been shown to influence the number of WPBs in endothelial cells, we propose to test two methods of attaining endothelial cell confluence to inform on the relevancy of cellular culture methods when analyzing endothelial WPBs. We test these cellular culture methods in two endothelial cell types, human umbilical vein endothelial cells (HUVECs) and endothelial colony forming cells (ECFCs). One method maintains a constant incubation time of 96 hrs. while varying the seeding density. The second method maintains a constant seeding density of 30,000 cells/cm2 while varying incubation time. In comparing these two methods, we evaluate the nuclei count, total WPB count, and WPB/nuclei count for each. Our results show that there is a trend of increasing nuclei count, total WPB count, and WPB/nuclei count as incubation time and seeding density increases. However, there is no difference in WPB/nuclei quantification whether confluency is reached via a constant seeding density or a constant incubation time. In addition, we show that confluency plays a major role in WPB/nuclei generation as we demonstrate higher WPB/nuclei counts in confluent cultures compared to sub-confluent cultures.

Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19. Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU. Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry. Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction. Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

Heterogeneity and reciprocity of FVIII and VWF expression, and the response to shear stress in cultured human endothelial cells

BackgroundSubstantial phenotypic heterogeneity exists in endothelial cells and while much of this heterogeneity results from local microenvironments, epigenetic modifications also contribute. MethodsCultured human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, human hepatic sinusoidal endothelial cells, human lymphatic endothelial cells (hLECs), and two different isolations of endothelial colony forming cells (ECFCs) were assessed for levels of factor VIII (FVIII) and von Willebrand factor (VWF) RNA and protein. The intracellular location and co‐localization of both proteins was evaluated with immunofluorescence microscopy and stimulated release toof FVIII and VWF from Weibel–Palade bodies (WPBs) was evaluated. Changes in expression of FVIII and VWF RNA after hLECs and ECFCs were exposed to 2 or 15 dynes/cm2 of laminar shear stress were also assessed. ResultsWe observed considerable heterogeneity in FVIII and VWF expression among the endothelial cells. With the exception of hLECs, FVIII RNA and protein were barely detectable in any of the endothelial cells and a reciprocal relationship between levels of FVIII and VWF appears to exist. When FVIII and VWF are co‐expressed, they do not consistently co‐localize in the cytoplasm. However, in hLECs where significantly higher levels of FVIII are expressed, FVIII and VWF co‐localize in WPBs and are released together when stimulated. Expression of both FVIII and VWF is markedly reduced when hLECs are exposed to higher or lower levels of laminar shear stress, while in ECFCs there is a minimal response for both proteins. ConclusionsVariable levels of FVIII and VWF RNA and protein exist in a subset of cultured human endothelial cells. Higher levels of FVIII present in hLECs co‐localize with VWF and are released together when exposed to a secretagogue.

Sustained VWF-ADAMTS-13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction

BackgroundProlonged recovery is common after acute SARS-CoV-2 infection; however, the pathophysiological mechanisms underpinning Long COVID syndrome remain unknown. VWF/ADAMTS-13 imbalance, dysregulated angiogenesis, and immunothrombosis are hallmarks of acute COVID-19. We hypothesized that VWF/ADAMTS-13 imbalance persists in convalescence together with endothelial cell (EC) activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation. Patients and methodsFifty patients were reviewed at a minimum of 6 weeks following acute COVID-19. ADAMTS-13, Weibel Palade Body (WPB) proteins, and angiogenesis-related proteins were assessed and clinical evaluation and immunophenotyping performed. Comparisons were made with healthy controls (n = 20) and acute COVID-19 patients (n = 36). ResultsADAMTS-13 levels were reduced (p = 0.009) and the VWF-ADAMTS-13 ratio was increased in convalescence (p = 0.0004). Levels of platelet factor 4 (PF4), a putative protector of VWF, were also elevated (p = 0.0001). A non-significant increase in WPB proteins Angiopoietin-2 (Ang-2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters. Enhanced expression of 21 angiogenesis-related proteins was observed in convalescent COVID-19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively. ConclusionOur data provide insights into sustained EC activation, dysregulated angiogenesis, and VWF/ADAMTS-13 axis imbalance in convalescent COVID-19. In keeping with the pivotal role of immunothrombosis in acute COVID-19, our findings support the hypothesis that abnormal T cell and monocyte populations may be important in the context of persistent EC activation and hemostatic dysfunction during convalescence.

Rabbit Endothelial Progenitor Cells Derived From Peripheral Blood and Bone Marrow: An Ultrastructural Comparative Study.

The present study was designed to compare the ultrastructure of early endothelial progenitor cells (EPCs) derived from rabbit peripheral blood (PB-EPCs) and bone marrow (BM-EPCs). After the cells had been isolated and cultivated up to passage 3, microphotographs obtained from transmission electron microscope were evaluated from qualitative and quantitative (unbiased stereological approaches) points of view. Our results revealed that both cell populations displayed almost identical ultrastructural characteristics represented by abundant cellular organelles dispersed in the cytoplasm. Moreover, the presence of very occasionally occurring mature endothelial-specific Weibel–Palade bodies (WPBs) confirmed their endothelial lineage origin. The more advanced stage of their differentiation was also demonstrated by the relatively low nucleus/cytoplasm (N/C) ratios (0.41 ± 0.19 in PB-EPCs; 0.37 ± 0.25 in BM-EPCs). Between PB-EPCs and BM-EPCs, no differences in proportions of cells occupied by nucleus (28.13 ± 8.97 versus 25.10 ± 11.48%), mitochondria (3.71 ± 1.33 versus 4.23 ± 1.00%), and lipid droplets (0.65 ± 1.01 versus 0.36 ± 0.40%), as well as in estimations of the organelles surface densities were found. The data provide the first quantitative evaluation of the organelles of interest in PB-EPCs and BM-EPCs, and they can serve as a research framework for understanding cellular function.

A Homozygous Deep Intronic Variant Causes Von Willebrand Factor Deficiency and Lack of Endothelial-Specific Secretory Organelles, Weibel-Palade Bodies.

A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of the conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promoter, exons, and flanking intronic regions of the VWF gene (VWF). In this study, we intended to elucidate causative mutation through next-generation sequencing (NGS) of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The NGS revealed a variant in the intronic region of VWF (997 + 118 T > G in intron 8), for the first time. The bioinformatics assessments (e.g., SpliceAl) predicted this variant creates a new donor splice site (ss), which could outcompete the consensus 5′ donor ss at exon/intron 8. This would lead to an aberrant mRNA that contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The subsequent quantitative real-time PCR confirmed the virtual absence of VWF mRNA in IP ECFCs. Additionally, the IP ECFCs demonstrated a considerable reduction in VWF secretion (~6% of healthy donors), and they were devoid of endothelial-specific secretory organelles, Weibel–Palade bodies. Our findings underline the potential of NGS in conjunction with RNA analysis and patient-derived cell studies for genetic diagnosis of mutation-negative type 3 VWD patients.

HPS6 Regulates the Biogenesis of Weibel-Palade Body in Endothelial Cells Through Trafficking v-ATPase to Its Limiting Membrane.

The Weibel–Palade body (WPB) is one of the lysosome-related organelles (LROs) in endothelial cells, whose main content is von Willebrand factor (vWF). The biogenesis of LROs is regulated by the Hermansky–Pudlak syndrome (HPS) protein-associated complexes through transporting cargo proteins to WPBs. Our previous studies have shown that HPS6, a subunit of BLOC-2 complex, is likely involved in the maturation of WPBs. However, the underlying mechanism remains unknown. In this study, we found that the knockdown of HPS6 in human umbilical vein endothelial cells (HUVECs) resulted in misshaped WPBs, decreased WPB number, and impaired vWF tubulation, which are similar to the characteristics of HPS6-deficient mouse endothelial cells. We observed similar morphological changes of WPBs in HUVECs after the knockdown of ATP6V0D1 (a subunit of v-ATPase). Furthermore, we found that HPS6 interacted with ATP6V0D1, suggesting that HPS6 transports ATP6V0D1 to the WPB limiting membrane for the assembly of the v-ATPase complex to maintain its acidic luminal pH, which is critical for the formation of vWF tubules during WPB maturation. In conclusion, HPS6 likely regulates the biogenesis of WPBs by participating in the trafficking of v-ATPase to the WPB membrane.

Single-cell transcriptional analysis of human endothelial colony-forming cells from patients with low VWF levels

Abstractvon Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.