Introduction: Traditional iron parameters often fail to distinguish the cause of iron-restricted anemia in patients without an obvious underlying cause. We evaluated whether an oral iron absorption... Show moreIntroduction: Traditional iron parameters often fail to distinguish the cause of iron-restricted anemia in patients without an obvious underlying cause. We evaluated whether an oral iron absorption test (OIAT) and hepcidin measurement could be useful diagnostic tests in these patients.Methods: We retrospectively analyzed data extracted from medical records of all patients who underwent an OIAT and hepcidin measurement, noting subsequent clinical diagnosis. Delta iron >15 mu mol/L during the OIAT and hepcidin level below the median (or suppressed <= 0.5 nM) were considered appropriate.Results: Thirty-nine adult patients were included in the study. Sixteen patients with adequate OIAT had suppressed hepcidin levels indicative of classical iron-deficiency anemia (IDA); 59% of patients had abnormal OIAT. In this group, most patients with low hepcidin levels had anemia associated with abnormalities in the gastrointestinal tract, whereas 83.3% patients with high hepcidin levels had iron-refractory iron deficiency anemia (IRIDA), confirmed by genetic testing. Finally, transferrin/log ferritin ratio accurately identified patients with suppressed hepcidin: AUC 0.98 [95% CI: 0.95-1.02], P < 0.001. Conclusion: OIAT differentiates between classical IDA and other types of anemia caused by abnormalities in iron absorption or systemic iron availability. Additionally, elevated hepcidin in patients with oral iron malabsorption could indicate IRIDA. Show less
An imbalance in von Willebrand factor (VWF) may either lead to bleeding (von Willebrand disease, VWD) or thrombosis. Both disorders have shortcomings in the currently available treatments. VWF... Show moreAn imbalance in von Willebrand factor (VWF) may either lead to bleeding (von Willebrand disease, VWD) or thrombosis. Both disorders have shortcomings in the currently available treatments. VWF itself could be a potential therapeutic target because of its role in both bleeding and thrombosis. Inhibiting VWF gene expression through allele-selective silencing of VWF with small interfering RNAs (siRNAs) could be a personalized approach to specifically inhibit mutant VWF in VWD or to normalize increased VWF levels in thrombotic disorders without complete VWF knockdown. Therefore, we investigated a method to allele-selectively silence the VWF gene in mice as a therapeutic strategy. Fourteen candidate siRNAs targeting murine Vwf of either the C57BL/6J (B6) or the 129S1/SvImJ (129S) strain were tested in vitro in cells expressing B6- and 129S-Vwf for inhibitory effect and allele-selective potential. Together with a nonselective siVwf, 2 lead candidate siRNAs, siVwf.B6 and siVwf.129S, were further tested in vivo in B6 and 129S mice. Efficient endothelial siRNA delivery was achieved by siRNA encapsulation into 7C1 oligomeric lipid nanoparticles. Treatment with the nonselective siVwf resulted in dose-dependent inhibition of up to 80% of both lung messenger RNA and plasma VWF protein in both mouse strains. In contrast, the allele-selective siVwf.B6 and siVwf.129S were shown to be effective in and selective solely for their corresponding mouse strain. To conclude, we showed efficient endothelial delivery of siRNAs that are highly effective in allele-selective inhibition of Vwf in mice, which constitutes an in vivo proof of principle of allele-selective VWF silencing as a therapeutic approach. Show less
Sier, V.Q.; Jong, A. de; Quax, P.H.A.; Vries, M.R. de 2022
Vein grafts (VGs) are used to bypass atherosclerotic obstructions and arteriovenous fistulas (AVFs) as vascular access for hemodialysis. Vascular remodeling governs post-interventional... Show moreVein grafts (VGs) are used to bypass atherosclerotic obstructions and arteriovenous fistulas (AVFs) as vascular access for hemodialysis. Vascular remodeling governs post-interventional arterialization, but may also induce VG and AVF failure. Although the endpoint characteristics of vascular remodeling are known, the in vivo process and the role of blood flow dynamics has not been fully studied. Therefore, here we non-invasively quantify vascular remodeling and blood flow alterations over time in murine VG and AVF models. C57BL/6J (n = 7, chow diet) and atherosclerosis-prone ApoE3*Leiden (n = 7) mice underwent VG surgery. Ultrasound imaging was performed at 3, 7, 14, 21, and 28 days post-surgery. C57BL/6J mice (n = 8) received AVF surgery. Ultrasound imaging was performed at 7 and 14 days post-surgery. The luminal volume increased by 42% in the VGs of C57BL/6J and 38% in the VGs of ApoE3*Leiden mice at 28 days relative to 3 days post-surgery. Longitudinally, an 82% increase in wall volume and 76% increase in outward remodeling was found in the ApoE3*Leiden mice, with a constant wall size in C57BL/6J mice. Proximally, the pulsatility index, resistive index, and peak systolic velocity decreased longitudinally in both groups. Distally, the maximum acceleration increased with 56% in C57BL/6J VGs. Among the AVFs, 50% showed maturation after 7 days, based on a novel flow-criterium of 23 mL/min. Distinct flow patterns were observed at the anastomotic site and inflow artery of the AVFs relative to the control carotid arteries. Vascular remodeling can be quantified by ultra-high-frequency ultrasound imaging over time in complex animal models, via three-dimensional structural parameters and site-specific hemodynamic indices. Show less
BACKGROUND: Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal... Show moreBACKGROUND: Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal hyperplasia, whereby endothelial cell dysfunction, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation play a crucial role. von Willebrand Factor (vWF) is an endothelial cell-derived protein involved in platelet aggregation and VSMC proliferation. We investigated AVF vascular remodeling in vWF-deficient mice and vWF expression in failed and matured human AVFs.METHODS AND RESULTS: Jugular-carotid AVFs were created in wild-type and vWF(-/-) mice. AVF flow was determined longitudinally using ultrasonography, whereupon AVFs were harvested 14 days after surgery. VSMCs were isolated from vena cavae to study the effect of vWF on VSMC proliferation. Patient-matched samples of the basilic vein were obtained before brachiobasilic AVF construction and during superficialization or salvage procedure 6 weeks after AVF creation. vWF deficiency reduced VSMC proliferation and macrophage infiltration in the intimal hyperplasia. vWF(-/-) mice showed reduced outward remodeling (1.5-fold, P=0.002) and intimal hyperplasia (10.2-fold, P<0.0001). AVF flow in wild-type mice was incremental over 2weeks, whereas flow in vWF(-/-) mice did not increase, resulting in a two-fold lower flow at 14 days compared with wild-type mice (P=0.016). Outward remodeling in matured patient AVFs coincided with increased local vWF expression in the media of the venous outflow tract. Absence of vWF in the intimal layer correlated with an increase in the intima-media ratio.CONCLUSIONS: vWF enhances AVF maturation because its positive effect on outward remodeling outweighs its stimulating effect on intimal hyperplasia. Show less
Fernandes, H.; Zonnari, A.; Abreu, R.; Aday, S.; Barao, M.; Albino, I.; ... ; Ferreira, L. 2022
Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is... Show moreEndothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5pmodulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocininduced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization. Show less
Jong, A. de; Sier, V.Q.; Peters, H.A.B.; Schilder, N.K.M.; Jukema, J.W.; Goumans, M.J.T.H.; ... ; Vries, M.R. de 2022
Aims: Vein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-beta via TGF beta... Show moreAims: Vein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-beta via TGF beta R2 or BMP9/10 via BMPR2, which is an important pathway in fibrotic, inflammatory, and angiogenic processes in vascular diseases. The role of the TGF-beta pathway in VGD is previously reported, however, the contribution of ALK1 signaling is not known. Therefore, we investigated ALK1 signaling in VGD in a mouse model for vein graft disease using either genetic or pharmacological inhibition of the Alk1 signaling. Methods and Results: Male ALK1 heterozygous (ALK1(+/-)), control C57BL/6, as well as hypercholesterolemic ApoE3*Leiden mice, underwent vein graft surgery. Histologic analyses of ALK1(+/-) vein grafts demonstrated increased outward remodeling and macrophage accumulation after 28 days. In hypercholesterolemic ApoE3*Leiden mice receiving weekly ALK1-Fc injections, ultrasound imaging showed 3-fold increased outward remodeling compared to controls treated with control-Fc, which was confirmed histologically. Moreover, ALK1-Fc treatment reduced collagen and smooth muscle cell accumulation, increased macrophages by 1.5-fold, and resulted in more plaque dissections. No difference was observed in intraplaque neovessel density. Flow cytometric analysis showed increased systemic levels of Ly6C(High) monocytes in ALK1-Fc treated mice, supported by in vitro increased MCP-1 and IL-6 production of LPS-stimulated and ALK1-Fc-treated murine monocytes and macrophages. Conclusion: Reduced ALK1 signaling in VGD promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype. Translational Perspective: Vein graft disease (VGD) severely hampers patency rates of vein grafts, necessitating research of key disease-driving pathways like TGF-beta. The three-dimensional nature of VGD together with the multitude of disease driving factors ask for a comprehensive approach. Here, we combined in vivo ultrasound imaging, histological analyses, and conventional in vitro analyses, identifying the ambiguous role of reduced ALK1 signaling in vein graft disease. Reduced ALK1 signaling promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype in murine vein grafts. Characterization of in vivo vascular remodeling over time is imperative to monitor VGD development and identify new therapies. Show less
Ingen, E. van; Foks, A.C.: Woudenberg, T.; Bent, M.L. van der; Jong, A. de; Hohensinner, P.J.; Wojta, J.; ... ; Nossent, A.Y. 2021
We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in... Show moreWe have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macrophage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened M1 polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability. Show less
Ingen, E. van; Foks, A.C.; Woudenberg, T.; Bent, M.L. van der; Jong, A. de; Hohensinner, P.J.; ... ; Nossent, A.Y. 2021
We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in... Show moreWe have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macro-phage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened Ml polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability. Show less
(1) Background: Vascular surgery operations are hampered by high failure rates and frequent occurrence of peri-operative cardiovascular complications. In pre-clinical studies, pre-operative... Show more(1) Background: Vascular surgery operations are hampered by high failure rates and frequent occurrence of peri-operative cardiovascular complications. In pre-clinical studies, pre-operative restriction of proteins and/or calories (PCR) has been shown to limit ischemia-reperfusion damage, slow intimal hyperplasia, and improve metabolic fitness. However, whether these dietary regimens are feasible and safe in the vascular surgery patient population remains unknown. (2) Methods: We performed a randomized controlled trial in patients scheduled for any elective open vascular procedure. Participants were randomized in a 3:2 ratio to either four days of outpatient pre-operative PCR (30% calorie, 70% protein restriction) or their regular ad-libitum diet. Blood was drawn at baseline, pre-operative, and post-operative day 1 timepoints. A leukocyte subset flow cytometry panel was performed at these timepoints. Subcutaneous/perivascular adipose tissue was sampled and analyzed. Follow-up was one year post-op. (3) Results: 19 patients were enrolled, of whom 11 completed the study. No diet-related reasons for non-completion were reported, and there was no intervention group crossover. The PCR diet induced weight loss and BMI decrease without malnutrition. Insulin sensitivity was improved after four days of PCR (p = 0.05). Between diet groups, there were similar rates of re-intervention, wound infection, and cardiovascular complications. Leukocyte populations were maintained after four days of PCR. (4) Conclusions: Pre-operative PCR is safe and feasible in elective vascular surgery patients. Show less
Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGF beta/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular... Show moreHereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGF beta/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/- mice, a model for HHT1. Eng+/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6C(high)/CD206(-)) at the expense of reparative M2-like macrophages (Ly6C(low)/CD206(+)). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGF beta-induced differentiation of Eng+/- monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/- mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/- mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/- mice. Show less
Background: Inflammatory stimuli induced by NF-kB drive atherosclerotic lesion formation. The epigenetic P300/CBP associated factor (PCAF) post-transcriptionally acetylates FoxP3, which is required... Show moreBackground: Inflammatory stimuli induced by NF-kB drive atherosclerotic lesion formation. The epigenetic P300/CBP associated factor (PCAF) post-transcriptionally acetylates FoxP3, which is required for regulatory T-cell (Treg) differentiation and immune modulation. We hypothesize that PCAF deficiency affects atherosclerosis via regulation of regulatory Tregs.Method: ApoE3*Leiden (n = 13) and ApoE3*LeidenxPCAF(-/-) (n = 13) were fed a high-fat diet (HFD) containing 1.25% cholesterol. Systemic FoxP3(+) T cells were measured every 4 weeks by flow cytometry (n = 6). After 5-months of HFD, mice were euthanized, and hearts and blood were collected. IL-6 and TNF alpha concentrations were measured in plasma to identify systemic inflammatory responses. Compositional and morphometrical analyses were performed on the atherosclerotic lesions in the aortic sinuses.Results: After 5 months of HFD, plasma cholesterol concentrations were not different for ApoE3*LeidenxPCAF(-/-) compared to ApoE3*Leiden mice. Expression of FoxP3 by systemic CD4(+) T cells decreased 1.8 fold in ApoE3*LeidenxPCAF(-/-) after 5 months HFD and remained significantly reduced after 5 months of HFD. Systemic TNF alpha and IL-6 concentrations were comparable, whereas the atherosclerotic lesion size in ApoE3*LeidenxPCAF(-/-) mice was increased by 28% compared to ApoE3*Leiden mice. In atherosclerotic lesions, no differences were observed in macrophage differentiation or VSMC content, although a small increase in collagen was identified.Conclusion: Our data show that PCAF deficiency resulted in a decrease in circulatory FoxP3(+) regulatory T cells and ameliorated atherosclerotic lesions with no differences in systemic inflammation or macrophage differentiation in the atherosclerotic lesions. This suggests that PCAF regulates atherosclerosis via modulation of FoxP3(+) regulatory T cell differentiation. Show less
Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and... Show morePrecise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB,n = 41) and bone marrow (BM;n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r(2) > 0.95 for all cell types in PB andr(2) > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures. Show less
Boer, S. de; Bowman, M.; Notley, C.; Mo, A.M.; Lima, P.; Jong, A. de; ... ; Eikenboom, J. 2020
Background Endothelial colony forming cells (ECFCs) derived from peripheral blood can be used to analyze the pathophysiology of vascular diseases ex vivo. However, heterogeneity is observed between... Show moreBackground Endothelial colony forming cells (ECFCs) derived from peripheral blood can be used to analyze the pathophysiology of vascular diseases ex vivo. However, heterogeneity is observed between ECFC clones and this variability needs to be understood and standardized for ECFCs to be used as a cell model for applications in vascular studies. Objective Determine reference characteristics of healthy control ECFCs to generate a valid ex vivo model for vascular disease. Methods Putative ECFCs (n = 47) derived from 21 individual healthy subjects were studied for cell morphology and specific cell characteristics. Clones were analyzed for the production and secretion of von Willebrand factor (VWF), cell proliferation, and the expression of endothelial cell markers. Results Based on morphology, clones were categorized into three groups. Group 1 consisted of clones with classic endothelial cell morphology, whereas groups 2 and 3 contained less condensed cells with increasing cell sizes. All clones had comparable endothelial cell surface expression profiles, with low levels of non-endothelial markers. However, a decrease in CD31 and a group-related increase in CD309 and CD45 expression, combined with a decrease in cell proliferation and VWF production and secretion, was observed in clones in group 3 and to a lesser extent in group 2. Conclusions We observed group-related variations in endothelial cell characteristics when clones lacked the classic endothelial cell morphology. Despite this variation, clones in all groups expressed endothelial cell surface markers. Provided that clones with similar characteristics are compared, we believe ECFCs are a valid ex vivo model to study vascular disease. Show less
Jong, A. de; Dirven, R.J.; Boender, J.; Atiq, F.; Anvar, S.Y.; Leebeek, F.W.G.; ... ; Eikenboom, J. 2020
Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These... Show moreVon Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequentVWFsingle nucleotide polymorphisms to inhibit mutantVWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes. Show less
Cartwright, A.; Webster, S.J.; Jong, A. de; Dirven, R.J.; Bloomer, L.D.S.; AL-Buhairan, A.M.; ... ; Willebrand Dis Study Grp von 2020
Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV... Show moreCopy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion. Show less
Baganha, F.; Jong, A. de; Jukema, J.W.; Quax, P.H.A.; Vries, M.R. de 2020
Obstructive arterial disease is a major cause of morbidity and mortality in the developed world. Venous bypass graft surgery is one of the most frequently used revascularization strategies despite... Show moreObstructive arterial disease is a major cause of morbidity and mortality in the developed world. Venous bypass graft surgery is one of the most frequently used revascularization strategies despite its considerable short and long time failure rate. Due to vessel wall remodeling, inflammation, intimal hyperplasia, and accelerated atherosclerosis, vein grafts may (ultimately) fail to revascularize tissues downstream to occlusive atherosclerotic lesions. In the past decades, little has changed in the prevention of vein graft failure (VGF) although new insights in the role of innate and adaptive immunity in VGF have emerged. In this review, we discuss the pathophysiological mechanisms underlying the development of VGF, emphasizing the role of immune response and associated factors related to VG remodeling and failure. Moreover, we discuss potential therapeutic options that can improve patency based on data from both preclinical studies and the latest clinical trials. This review contributes to the insights in the role of immunomodulation in vein graft failure in humans. We describe the effects of immune cells and related factors in early (thrombosis), intermediate (inward remodeling and intimal hyperplasia), and late (intimal hyperplasia and accelerated atherosclerosis) failure based on both preclinical (mouse) models and clinical data. Show less
Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected... Show moreVon Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected patients may also develop joint bleeding, or bleeding from the gastrointestinal tract. Also, trauma, surgery or dental procedures may lead to critical bleeding events. VWD-related bleeding are caused by defects in von Willebrand factor (VWF), a large multimeric protein that is produced by endothelial cells and megakaryocytes. Most VWD patients develop the disease because of dominant-negative mutations in VWF. In this thesis we investigated whether inhibition of production of mutant VWF with limited inhibition of wildtype VWF positively affects the function of VWF and improves VWD phenotypes. We used small interfering RNAs (siRNAs) to selectively inhibit production of mutant VWF. These siRNAs were tested in several models for VWD. We indeed prove that siRNAs can distinguish a mutant and wildtype VWF allele in vitro in heterologous cell systems, ex vivo in patient-derived endothelial cells, and in vivo in a VWD mouse model. We also show in these disease models that we can improve several VWD phenotypes. These results are promising for further development of allele-specific siRNAs as a new treatment strategy for VWD. Show less
Stahle, M.; Silvola, J.M.U.; Hellberg, S.; Vries, M. de; Quax, P.H.A.; Kroon, J.; ... ; Saraste, A. 2020
This study showed that treatment with a therapeutic monoclonal immunoglobutin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates... Show moreThis study showed that treatment with a therapeutic monoclonal immunoglobutin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of F-18-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies. (C) 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. Show less
