FLOW: Flow dysfunction of hemodialysis vascular access: A randomized controlled trial on the effectiveness of surveillance of arteriovenous fistulas and grafts.

INTRODUCTION: It is assumed that identification and correction of asymptomatic stenoses in the vascular access circuit will prevent thrombosis that would require urgent intervention to continue hemodialysis treatment. However, the evidence base for this assumption is limited. Recent international clinical practice guidelines reach different conclusions on the use of surveillance for vascular access flow dysfunction and recommend further research to inform clinical practice. METHODS: The FLOW trial is a double-blind, multicenter, randomized controlled trial with a 1:1 individual participant treatment allocation ratio over two study arms. In the intervention group, only symptomatic vascular access stenoses detected by clinical monitoring are treated, whereas in the comparison group asymptomatic stenoses detected by surveillance using monthly dilution flow measurements are treated as well. Hemodialysis patients with a functional arteriovenous vascular access are enrolled. The primary outcome is the access-related intervention rate that will be analyzed using a general linear model with Poisson distribution. Secondary outcomes include patient satisfaction, access-related serious adverse events, and quality of the surveillance process. A cost effectiveness analysis and budget impact analysis will also be conducted. The study requires 828 patient-years of follow-up in 417 participants to detect a difference of 0.25 access-related interventions per year between study groups. DISCUSSION: As one of the largest randomized controlled trials assessing the clinical impact of vascular access surveillance using a strong double-blinded study design, we believe the FLOW trial will provide much-needed evidence to improve vascular access care for hemodialysis patients.

Mechanical Strain Induces Transcriptomic Reprogramming of Saphenous Vein Progenitors.

Intimal hyperplasia is the leading cause of graft failure in aortocoronary bypass grafts performed using human saphenous vein (SV). The long-term consequences of the altered pulsatile stress on the cells that populate the vein wall remains elusive, particularly the effects on saphenous vein progenitors (SVPs), cells resident in the vein adventitia with a relatively wide differentiation capacity. In the present study, we performed global transcriptomic profiling of SVPs undergoing uniaxial cyclic strain in vitro. This type of mechanical stimulation is indeed involved in the pathology of the SV. Results showed a consistent stretch-dependent gene regulation in cyclically strained SVPs vs. controls, especially at 72 h. We also observed a robust mechanically related overexpression of Adhesion Molecule with Ig Like Domain 2 (AMIGO2), a cell surface type I transmembrane protein involved in cell adhesion. The overexpression of AMIGO2 in stretched SVPs was associated with the activation of the transforming growth factor ß pathway and modulation of intercellular signaling, cell-cell, and cell-matrix interactions. Moreover, the increased number of cells expressing AMIGO2 detected in porcine SV adventitia using an in vivo arterialization model confirms the upregulation of AMIGO2 protein by the arterial-like environment. These results show that mechanical stress promotes SVPs' molecular phenotypic switching and increases their responsiveness to extracellular environment alterations, thus prompting the targeting of new molecular effectors to improve the outcome of bypass graft procedure.

Coronary artery mechanics induces human saphenous vein remodelling via recruitment of adventitial myofibroblast-like cells mediated by Thrombospondin-1.

Rationale: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease. Methods: We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results in vitro and using a model of SV carotid interposition in pigs. Results: Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22α) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-ß-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-ß-dependent pathway, and validated in a saphenous vein into carotid interposition pig model. Conclusions: Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors.

Versican is differentially regulated in the adventitial and medial layers of human vein grafts.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation.

Long-term effect of stents eluting 6-mercaptopurine in porcine coronary arteries.

BACKGROUND: Drug-eluting stents (DES) have dramatically reduced restenosis rates compared to bare metal stents and are widely used in coronary artery angioplasty. The anti-proliferative nature of the drugs reduces smooth muscle cell (SMC) proliferation effectively, but unfortunately also negatively affects endothelialization of stent struts, necessitating prolonged dual anti-platelet therapy. Cell-type specific therapy may prevent this complication, giving rise to safer stents that do not require additional medication. 6-Mercaptopurine (6-MP) is a drug with demonstrated cell-type specific effects on vascular cells both in vitro and in vivo, inhibiting proliferation of SMCs while promoting survival of endothelial cells. In rabbits, we demonstrated that DES locally releasing 6-MP during 4 weeks reduced in-stent stenosis by inhibiting SMC proliferation and reducing inflammation, without negatively affecting endothelialization of the stent surface. The aim of the present study was to investigate whether 6-MP-eluting stents are similarly effective in preventing stenosis in porcine coronary arteries after 3 months, in order to assess the eligibility for human application. METHODS: 6-MP-eluting and polymer-only control stents (both n = 7) were implanted in porcine coronary arteries after local balloon injury to assess the effect of 6-MP on vascular lesion formation. Three months after implantation, stented coronary arteries were harvested and analyzed. RESULTS: Morphometric analyses revealed that stents were implanted reproducibly and with limited injury to the vessel wall. Unexpectedly, both in-stent stenosis (6-MP: 41.1 ± 10.3 %; control: 29.6 ± 5.9 %) and inflammation (6-MP: 2.14 ± 0.51; control: 1.43 ± 0.45) were similar between the groups after 3 months. CONCLUSION: In conclusion, although 6-MP was previously found to potently inhibit SMC proliferation, reduce inflammation and promote endothelial cell survival, thereby effectively reducing in-stent restenosis in rabbits, stents containing 300 µg 6-MP did not reduce stenosis and inflammation in porcine coronary arteries.

Stents Eluting 6-Mercaptopurine Reduce Neointima Formation and Inflammation while Enhancing Strut Coverage in Rabbits.

BACKGROUND: The introduction of drug-eluting stents (DES) has dramatically reduced restenosis rates compared with bare metal stents, but in-stent thrombosis remains a safety concern, necessitating prolonged dual anti-platelet therapy. The drug 6-Mercaptopurine (6-MP) has been shown to have beneficial effects in a cell-specific fashion on smooth muscle cells (SMC), endothelial cells and macrophages. We generated and analyzed a novel bioresorbable polymer coated DES, releasing 6-MP into the vessel wall, to reduce restenosis by inhibiting SMC proliferation and decreasing inflammation, without negatively affecting endothelialization of the stent surface. METHODS: Stents spray-coated with a bioresorbable polymer containing 0, 30 or 300 µg 6-MP were implanted in the iliac arteries of 17 male New Zealand White rabbits. Animals were euthanized for stent harvest 1 week after implantation for evaluation of cellular stent coverage and after 4 weeks for morphometric analyses of the lesions. RESULTS: Four weeks after implantation, the high dose of 6-MP attenuated restenosis with 16% compared to controls. Reduced neointima formation could at least partly be explained by an almost 2-fold induction of the cell cycle inhibiting kinase p27Kip1. Additionally, inflammation score, the quantification of RAM11-positive cells in the vessel wall, was significantly reduced in the high dose group with 23% compared to the control group. Evaluation with scanning electron microscopy showed 6-MP did not inhibit strut coverage 1 week after implantation. CONCLUSION: We demonstrate that novel stents coated with a bioresorbable polymer coating eluting 6-MP inhibit restenosis and attenuate inflammation, while stimulating endothelial coverage. The 6-MP-eluting stents demonstrate that inhibition of restenosis without leaving uncovered metal is feasible, bringing stents without risk of late thrombosis one step closer to the patient.

Inhibition of GTPase Rac1 in endothelium by 6-mercaptopurine results in immunosuppression in nonimmune cells: new target for an old drug.

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory diseases.

Diabetes impairs arteriogenesis in the peripheral circulation: review of molecular mechanisms.

Patients suffering from both diabetes and PAD (peripheral arterial disease) are at risk of developing critical limb ischaemia and ulceration, and potentially requiring limb amputation. In addition, diabetes complicates surgical treatment of PAD and impairs arteriogenesis. Arteriogenesis is defined as the remodelling of pre-existing arterioles into conductance vessels to restore the perfusion distal to the occluded artery. Several strategies to promote arteriogenesis in the peripheral circulation have been devised, but the mechanisms through which diabetes impairs arteriogenesis are poorly understood. The present review provides an overview of the current literature on the deteriorating effects of diabetes on the key players in the arteriogenesis process. Diabetes affects arteriogenesis at a number of levels. First, it elevates vasomotor tone and attenuates sensing of shear stress and the response to vasodilatory stimuli, reducing the recruitment and dilatation of collateral arteries. Secondly, diabetes impairs the downstream signalling of monocytes, without decreasing monocyte attraction. In addition, EPC (endothelial progenitor cell) function is attenuated in diabetes. There is ample evidence that growth factor signalling is impaired in diabetic arteriogenesis. Although these defects could be restored in animal experiments, clinical results have been disappointing. Furthermore, the diabetes-induced impairment of eNOS (endothelial NO synthase) strongly affects outward remodelling, as NO signalling plays a key role in several remodelling processes. Finally, in the structural phase of arteriogenesis, diabetes impairs matrix turnover, smooth muscle cell proliferation and fibroblast migration. The review concludes with suggestions for new and more sophisticated therapeutic approaches for the diabetic population.

Impaired collateral recruitment and outward remodeling in experimental diabetes.

OBJECTIVE: In this study, the effect of chronic hyperglycemia on acute ligation-induced collateral vasodilation, on monocyte chemotaxis, and on structural outward remodeling of collaterals was investigated. RESEARCH DESIGN AND METHODS: Femoral artery ligation was performed 8 weeks after alloxan or saline treatment in New Zealand White rabbits. Angiography was performed directly, 1 and 3 weeks after ligation. These angiographic recordings were used to quantify number of collaterals, lumen, and blood volume index. Reactive hyperemia response was tested by intramuscular laser Doppler measurements. Subsequently, blood was sampled from the aorta for monocyte chemotaxis. RESULTS: Ligation resulted in markedly lower acute collateral vasodilation in diabetic compared with control rabbits. Also, hyperemic vasodilatory response to local ischemia was impaired in diabetic rabbits. This difference persisted at 1 and 3 weeks after ligation, with a lower number of visible collaterals. In addition, the collateral lumen was markedly lower in diabetic rabbits after the maturation phase. Likewise, a reduced blood volume index in the region of growing collaterals was observed in diabetic animals. The monocyte migration toward vascular endothelial growth factor-A and monocyte chemotactic protein-1 was strongly reduced in diabetic rabbits. CONCLUSIONS: This study demonstrates that chronic hyperglycemia negatively affects the different phases of arteriogenesis: 1) impaired shear induced vasodilatation; 2) impaired outward collateral growth, reflected in the number of collaterals and blood volume index; and 3) inhibition of monocyte chemotaxis. Impairments were most evident in the acute phase of arteriogenesis. Therapies aimed at restoring acute collateral recruitment, such as vasodilators, may be of interest to improve collateral function in diabetes.