Daniel Eefting

Wnt but not BMP signaling is involved in the inhibitory action of sclerostin on BMP-stimulated bone formation

Sclerostin is an osteocyte‐derived negative regulator of bone formation. It inhibits BMP‐stimulated bone formation both in vitro and in vivo but has no direct effect on BMP signaling. Instead, sclerostin inhibits Wnt signaling that is required for BMP‐stimulated osteoblastic differentiation.

Anti–MCP-1 Gene Therapy Inhibits Vascular Smooth Muscle Cells Proliferation and Attenuates Vein Graft Thickening Both In Vitro and In Vivo

Objective—Because late vein graft failure is caused by intimal hyperplasia (IH) and accelerated atherosclerosis, and these processes are thought to be inflammation driven, influx of monocytes is one of the first phenomena seen in IH, we would like to provide direct evidence for a role of the MCP-1 pathway in the development of vein graft disease. Methods and Results—MCP-1 expression is demonstrated in various stages of vein graft disease in a murine model in which venous interpositions are placed in the carotid arteries of hypercholesterolemic ApoE3Leiden mice and in cultured human saphenous vein (HSV) segments in which IH occurs. The functional involvement of MCP-1 in vein graft remodeling is demonstrated by blocking the MCP-1 receptor CCR-2 using 7ND-MCP-1. 7ND-MCP1 gene transfer resulted in 51% reduction in IH in the mouse model, when compared with controls. In HSV cultures neointima formation was inhibited by 53%. In addition, we demonstrate a direct inhibitory effect of 7ND-MCP-1 on the proliferation of smooth muscle cell (SMC) in HSV cultures and in SMC cell cultures. Conclusion—These data, for the first time, prove that MCP-1 has a pivotal role in vein graft thickening due to intimal hyperplasia and accelerated atherosclerosis.

Natural Killer Cells and CD4 + T-Cells Modulate Collateral Artery Development

Objective—The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. Here, we studied the role of lymphocytes in a murine model of hindlimb ischemia. Methods and Results—Lymphocytes, detected with markers for NK1.1, CD3, and CD4, invaded the collateral vessel wall. Arteriogenesis was impaired in C57BL/6 mice depleted for Natural Killer (NK)-cells by anti-NK1.1 antibodies and in NK-cell–deficient transgenic mice. Arteriogenesis was, however, unaffected in J&agr;281-knockout mice that lack NK1.1+ Natural Killer T (NKT)-cells, indicating that NK-cells, rather than NKT-cells, are involved in arteriogenesis. Furthermore, arteriogenesis was impaired in C57BL/6 mice depleted for CD4+ T-lymphocytes by anti-CD4 antibodies, and in major histocompatibility complex (MHC)-class-II–deficient mice that more selectively lack mature peripheral CD4+ T-lymphocytes. This impairment was even more profound in anti-NK1.1-treated MHC-class-II–deficient mice that lack both NK- and CD4+ T-lymphocytes. Finally, collateral growth was severely reduced in BALB/c as compared with C57BL/6 mice, 2 strains with different bias in immune responsiveness. Conclusions—These data show that both NK-cells and CD4+ T-cells modulate arteriogenesis. Promoting lymphocyte activation may represent a promising method to treat ischemic disease.

Sirolimus and paclitaxel provoke different vascular pathological responses after local delivery in a murine model for restenosis on underlying atherosclerotic arteries

Background: Drug-eluting stents (DES) have been introduced successfully in clinical practice to prevent post-angioplasty restenosis. Nevertheless, concerns about the safety of DES still exist. Objective: To investigate the vascular pathology and transcriptional responses to sirolimus and paclitaxel in a murine model for restenosis on underlying diseased atherosclerotic arteries. Methods: Atherosclerotic lesions were induced by placement of a perivascular cuff around the femoral artery of hypercholesterolaemic ApoE*3-Leiden transgenic mice. Two weeks later these cuffs were replaced either by sirolimus- or paclitaxel-eluting cuffs. The vascular pathological effects were evaluated after two additional weeks. Results: Both anti-restenotic compounds significantly inhibited restenotic lesion progression on the atherosclerotic plaques. Vascular histopathological analyses showed that local delivery of sirolimus has no significant adverse effects on vascular disease. Conversely, high dosages of paclitaxel significantly increased apoptosis, internal elastic lamina disruption, and decreased medial and intimal smooth muscle cells and collagen content. Moreover, transcriptional analysis by real-time RT-PCR showed an increased level of pro-apoptotic mRNA transcripts (FAS, BAX, caspase 3) in paclitaxel-treated arteries. Conclusions: Sirolimus and paclitaxel are effective in preventing restenosis. Sirolimus has no significant effect on arterial disease. In contrast, paclitaxel at high concentration demonstrated adverse vascular pathology and transcriptional responses, suggesting a narrower therapeutic range of this potent drug. Since the use of overlapping stents is becoming more common in DES technology, this factor is important, given that higher dosages of paclitaxel may lead to increased apoptosis in the vessel wall and, consequently, to a more unstable phenotype of the pre-existing atherosclerotic lesion.

Interleukin 10: A new risk marker for the development of restenosis after percutaneous coronary intervention

Genetic factors appear to be important in the process of restenosis after percutaneous coronary intervention (PCI), as well as in inflammation, a pivotal factor in restenosis. An important mediator in the inflammatory response is interleukin (IL)-10. Our aim was to study whether genetic variants in IL-10 predispose to the risk of restenosis. The GENetic DEterminants of Restenosis (GENDER) study included 3104 patients treated with successful PCI. Target vessel revascularization (TVR) was chosen as primary end point. Genotyping of the −2849G/A, −1082G/A, −592C/A and +4259A/G polymorphisms of the IL-10 gene was performed by MassArray platform. After adjusting for clinical variables, three polymorphisms significantly increased the risk of restenosis (−2849AA: relative risk (RR), 1.7, 95% confidence interval (CI), 1.2–2.5; −1082AA: RR, 1.4, 95% CI, 1.1–1.8 and +4259GG: RR, 2.0, 95% CI, 1.4–2.8). To further exclude possible involvement of neighboring genes due to LD in the IL-10 locus, additional polymorphisms were genotyped. The results reveal that association of the IL-10 gene with restenosis is independent of flanking genes. Our findings demonstrate that IL-10 is associated with restenosis and therefore support the hypothesis that anti-inflammatory genes also may be involved in developing restenosis. Furthermore, they may provide a new targeting gene for drug-eluting stents.

Experience with the GORE EXCLUDER iliac branch endoprosthesis for common iliac artery aneurysms

OBJECTIVEnIn this study, we analyzed the procedural success and early outcome of endovascular treatment of a multicenter cohort of patients with common iliac artery (CIA) aneurysms treated with the new GORE EXCLUDER (W. L. Gore & Associates, Flagstaff, Ariz) iliac branch endoprosthesis (IBE).nnnMETHODSnA retrospective cohort analysis was performed in 13 sites in The Netherlands. Anatomic, demographic, procedural, and follow-up data were assessed from hospital records.nnnRESULTSnFrom November 2013 to December 2014, 51 CIA aneurysms were treated with an IBE in 46 patients. Thexa0median diameter of the treated aneurysm was 40.5 (range, 25.0-90.0) mm. The mean procedural time was 198xa0±xa056xa0minutes. All but one implantation were successful; two type Ib endoleaks were noticed, resulting in a procedural success rate of 93.5%. The two type Ib endoleaks spontaneously disappeared at 30xa0days. There was no 30-day mortality. Ipsilateral buttock claudication was present in only two cases at 30xa0days and disappeared during follow-up. The incidence of reported erectile dysfunction was low and severe ischemic complications were absent. After a mean follow-up of 6xa0months, data on 17 treated aneurysms were available. Two showed a stable diameter, whereas 15 showed a mean decrease of 3.9xa0± 2.2xa0mmxa0(Pxa0< .001). Reinterventions were performed in two patients (7.1%). The 6-month primary patency of the internal component of the IBE device was 94%.nnnCONCLUSIONSnThe use of the GORE EXCLUDER IBE device for CIA aneurysms is related to high procedural success, high patency rates, and low reintervention rates at short-term follow-up. Prospective data with longer follow-up are awaited to establish the role of the device in the treatment algorithm of CIA aneurysms.

A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease

AIMSnMatrix metalloproteinases (MMP) and plasminogen activator (PA)/plasmin-mediated proteolysis, especially at the cell surface, play important roles in matrix degeneration and smooth muscle cell migration, which largely contributes to vein graft failure. In this study, a novel hybrid protein was designed to inhibit both protease systems simultaneously. MMP and plasmin activity were inhibited at the cell surface by this hybrid protein, consisting of the receptor-binding amino-terminal fragment (ATF) of urokinase-type PA, linked to both the tissue inhibitor of metalloproteinases (TIMP-1) and bovine pancreas trypsin inhibitor (BPTI), a potent protease inhibitor. The effect of overexpression of this protein on vein graft disease was studied.nnnMETHODS AND RESULTSnA non-viral expression vector encoding the hybrid protein TIMP-1.ATF.BPTI was constructed and validated. Next, cultured segments of human veins were transfected with this vector. Expressing TIMP-1.ATF.BPTI in vein segments resulted in a mean 36 ± 14% reduction in neointima formation after 4 weeks. In vivo inhibition of vein graft disease by TIMP-1.ATF.BPTI is demonstrated in venous interpositions placed into carotid arteries of hypercholesterolaemic APOE*3Leiden mice. After 4 weeks, vein graft thickening was significantly inhibited in mice treated with the domains TIMP-1, ATF, or BPTI (36-49% reduction). In the TIMP-1.ATF.BPTI-treated mice, vein graft thickening was reduced by 67±4%, which was also significantly stronger when compared with the individual components.nnnCONCLUSIONnThese data provide evidence that cell surface-bound inhibition of the PA and MMP system by the hybrid protein TIMP-1.ATF.BPTI, overexpressed in distant tissues after electroporation-mediated non-viral gene transfer, is a powerful approach to prevent vein graft disease.

In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor.

BACKGROUNDnSmooth muscle cell (SMC) migration and proliferation are important in the development of intimal hyperplasia, the major cause of vein graft failure. Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system are pivotal in extracellular matrix degradation and, by that, SMC migration. Previously, we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the receptor-binding amino terminal fragment (ATF) of urokinase, reduces SMC migration and neointima formation in an in vitro restenosis model using human saphenous vein cultures more efficiently than both protease systems separately. Because use of viral gene delivery is difficult in clinical application, this study used nonviral delivery of TIMP-1.ATF plasmid to reduce vein graft disease in a murine bypass model. Nonviral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery, such as immune responses and short-term expression.nnnMETHODSnPlasmids encoding ATF, TIMP-1, TIMP-1.ATF, or luciferase, as a control, were injected and electroporated in both calf muscles of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3Leiden) mice (n = 8). One day after electroporation, a venous interposition of a donor mouse was placed into the carotid artery of a recipient mouse. In this model, vein graft thickening develops with features of accelerated atherosclerosis. Vein grafts were harvested 4 weeks after electroporation and surgery, and histologic analysis of the vessel wall was performed.nnnRESULTSnElectroporation-mediated overexpression of the plasmid vectors resulted in a prolonged expression of the transgenes and resulted in a significant reduction of vein graft thickening (ATF: 36% +/- 9%, TIMP-1: 49% +/- 5%, TIMP-1.ATF: 58% +/- 5%; P < .025). Although all constructs reduced vein graft thickening compared with the controls, the luminal area was best preserved in the TIMP-1.ATF-treated mice.nnnCONCLUSIONnIntramuscular electroporation of TIMP-1.ATF inhibits vein graft thickening in vein grafts in carotid arteries of hypercholesterolemic mice. Binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface enhances the inhibitory effect of TIMP-1 on vein graft remodeling in vitro as well as in vivo and may be an effective strategy to prevent vein graft disease.

Gore Iliac Branch Endoprosthesis for treatment of bilateral common iliac artery aneurysms

Objective: The Gore Iliac Branch Endoprosthesis (IBE; W. L. Gore & Associates, Flagstaff, Ariz) has recently been approved by the Food and Drug Administration for treatment of common iliac artery (CIA) aneurysms. Despite early excellent results in clinical trial, none of 63 patients were treated for bilateral iliac aneurysms. The goal of this study was to examine real‐world experience using the Gore IBE for bilateral CIA aneurysms. Methods: A retrospective review of an international multicenter (16 U.S., 8 European) experience using the Gore IBE to treat bilateral CIA aneurysms was performed. Cases were limited to those occurring after Food and Drug Administration approval (February 2016) in the United States and after CE mark approval (November 2013) in Europe. Demographics of the patients, presentation, anatomic characteristics, and procedural details were captured. Results: There were 47 patients (45 men; mean age, 68 years; range, 41‐84 years) treated with bilateral Gore IBEs (27 U.S., 20 European). Six patients (12.7%) were symptomatic and 12 (25.5%) patients were treated primarily for CIA aneurysm (aorta <5.0 cm). Mean CIA diameter was 40.3 mm. Four patients had aneurysmal internal iliac arteries (IIAs). Two of these were sealed proximally at the IIA aneurysm neck and two required coil embolization of IIA branches to achieve seal in the largest first‐order branches. Technical success was achieved in 46 patients (97.9%). No type I or type III endoleaks were noted. There was no significant perioperative morbidity or mortality. IIA branch adjunctive stenting was required in four patients (one IIA distal dissection, three kinks). On follow‐up imaging available for 40 patients (85.1%; mean, 6.5 months; range, 1‐36 months), 12 type II endoleaks (30%) and no type I or type III endoleaks were detected. Two of 80 (2.5%) IIA branches imaged were occluded; one was intentionally sacrificed perioperatively. Conclusions: Preservation of bilateral IIAs in repair of bilateral CIA aneurysms can be performed safely with excellent technical success and short‐term patency rates using the Gore IBE device. Limb and branch occlusions are rare, usually are due to kinking, and can almost always be treated successfully with stenting.

Supervised preoperative forearm exercise to increase blood vessel diameter in patients requiring an arteriovenous access for hemodialysis: rationale and design of the PINCH trial

Background: Failure of maturation occurs in 30%-60% of arteriovenous fistula (AVF) creation for hemodialysis, with highest rates in distal radiocephalic fistulas. This is partly due to initial small blood vessel size with limited blood flow capacity. Forearm exercise has shown potential as stimulus for increasing blood vessel size in patients with end-stage renal disease (ESRD) and may promote maturation of AVFs in the upper limb when applied postoperatively. However, it is unknown if forearm exercise increases blood vessel size pre-operatively, which may contribute to more distal AVF creation and raise success rates of AVF surgery. This study will investigate these issues. Methods and results: The PINCH trial (refers to ‘pinching’) is an investigator-initiated, multicenter, single-blinded, randomized controlled trial with 1:1 randomization to perform supervised forearm exercises compared to no exercise 6 weeks pre-operatively before creation of an AVF. Forty patients receiving an AVF will be included. The main study endpoints are blood vessel diameter (cephalic or basilic vein and radial and ulnar artery), AVF surgical plan (radiocephalic or brachiobasilic/cephalic), and three-month (assisted) maturation rate. The burden of the performed forearm exercises will be evaluated using Kidney Disease Quality of Life (KDQOL-SF Dutch version 1.2) and exercise specific questionnaires. The PINCH trial is planned to start in November 2017. Enrollment is expected to be completed at the end of 2019. Conclusions: The PINCH study is the first trial to evaluate the effect of pre-operative, supervised forearm exercises on vein diameter and fistula maturation in hemodialysis patients. Trial registration: NTR6382.