Fat emulsions based on structured lipids (1,3-specific triglycerides): an investigation of the in vitro interaction with plasma proteins

Structured lipids (1,3-specific triglycerides) are new chemical entities made by enzymatic transesterification of the fatty acids in the 1,3-positions of the triglyceride. The purpose of this study was to investigate the in vitro interaction of fat emulsions based on either structured lipids or vegetable oils with human plasma proteins employing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The structured lipids are triglycerides of the SLS and MLM types, where S is short-chain fatty acids (C4), M is medium-chain fatty acids (C8-10) and L is long-chain fatty acids (C16-18). The vegetable oil-based fat emulsions were the commercially available product, Intralipid, and a soybean oil (LLL) emulsion made de novo identically as the emulsions containing structured lipids. The SLS emulsion was found to adsorb a different protein pattern than the MLM and LLL emulsions. The protein pattern of the SLS emulsion was similar to the protein pattern of Intralipid. These findings might explain the in vivo difference in elimination found in another study, where the emulsion based on structured lipids with short-chain fatty acids in the 1,3-positions was removed more slowly from the general blood circulation compared to emulsions based on lipids with long-chain fatty acids in the 1,3-positions (LLL).     

Thrombomodulin activity of fat-derived microvascular endothelial cells seeded on expanded polytetrafluorethylene

Lining the luminal surface of prosthetic vascular grafts with endothelial cells (cell seeding) will lower its thrombogenicity. Commonly used macrovascular human adult endothelial cells (HAEC) require in vitro cultivation before large enough numbers are obtained to cover grafts confluently. Fat-derived microvascular endothelial cells (MVEC) prove to be a good alternative as they can be harvested in much larger numbers while showing similar antithrombotic and fibrinolytic characteristics. An important anticoagulant function of macrovascular endothelial cells is due to the activity of thrombomodulin (TM) on their surface. In this study, the presence and functional activity of TM on fat-derived microvascular cells used in cell seeding was investigated. The expression and localization of TM on MVEC was studied using immunohistochemistry. Functional activity of TM on MVEC was measured by the generation of activated protein C (APC) and was compared to human umbilical vein endothelial cells (HUVEC). TM activity was studied in MVEC seeded on expanded polytetrafluorethylene (ePTFE) vascular prostheses and compared to blank prostheses. We found that TM was expressed on the surface of MVEC, both in vivo and vitro. TM-dependent generation of APC differed significantly between MVEC and HUVEC (3.98 +/- 1.2 vs. 3.0 +/- 0.7 nM, respectively). After seeding MVEC on vascular prostheses, TM activity did not change. APC generation was significantly higher on MVEC-seeded vascular grafts compared to blank grafts (4.0 +/- 0.7 vs. 1.7 +/- 0.5 nM, respectively). We conclude that TM is present and highly active on cultured MVEC. When seeded on ePTFE, MVEC retain the possibility to inhibit thrombin coagulant activity and to activate protein C. Therefore, since MVEC are readily available, the anticoagulant properties demonstrated here indicate that this cell type is suitable for cell seeding of vascular prostheses.     

A systematic review and meta-analysis of the use of resuscitative endovascular balloon occlusion of the aorta in the management of major exsanguination

Background

Circulatory collapse is a leading cause of mortality among traumatic major exsanguination and in ruptured aortic aneurysm patients. Approximately 40% of patients die before hemorrhage control is achieved. Resuscitative endovascular balloon occlusion of the aorta (REBOA) is an adjunct designed to sustain the circulation until definitive surgical or endovascular repair. A systematic review was conducted for the current clinical use of REBOA in patients with hemodynamic instability and to discuss its potential role in improving prehospital and in-hospital outcome.

Methods

Systematic review and meta-analysis (1900–2017) using MEDLINE, Cochrane, EMBASE, Web of Science and Central and Emcare using the keywords “aortic balloon occlusion”, “aortic balloon tamponade”, “REBOA”, and “Resuscitative Endovascular Balloon Occlusion” in combination with hemorrhage control, hemorrhage, resuscitation, shock, ruptured abdominal or thoracic aorta, endovascular repair, and open repair. Original published studies on human subjects were considered.

Results

A total of 490 studies were identified; 89 met criteria for inclusion. Of the 1436 patients, overall reported mortality was 49.2% (613/1246) with significant differences (p?<?0.001) between clinical indications. Hemodynamic shock was evident in 79.3%, values between clinical indications showed significant difference (p?<?0.001). REBOA was favored as treatment in trauma patients in terms of mortality. Pooled analysis demonstrated an increase in mean systolic pressure by almost 50 mmHg following REBOA use.

Conclusion

REBOA has been used in trauma patients and ruptured aortic aneurysm patients with improvement of hemodynamic parameters and outcomes for several decades. Formal, prospective study is warranted to clarify the role of this adjunct in all hemodynamic unstable patients.

     

Implementation of Endovenous Laser Ablation for Varicose Veins in a Large Community Hospital: The First 400 Procedures

Endovenous laser ablation (ELA) has become a standard treatment of the incompetent great saphenous vein (GSV). Our prospective audit examines the implementation of this new method in a large community hospital with special attention to obstacles, technical results, pain scores, failures and our learning curve.MethodsThree hundred and twenty-three patients (403 limbs) with incompetence of the GSV underwent ELA. Patients were assessed by clinical examination and venous duplex ultrasound was performed 6 weeks after operation. Visual analog scale (VAS) pain scores of the first postoperative week were recorded. Operative time and success rate were analysed.ResultsAfter 6 weeks, 301 (74.7%) treated legs were examined by duplex ultrasound imaging. Successful complete occlusion was present in 282 (93.7%) GSVs. Partial occlusion was present in 12 (4.0%) GSVs. In seven (2.3%) limbs the GSV was not occluded. The maximum mean VAS pain score was noted on the 5th postoperative day. From the start of this series, the operation time decreased rapidly for each surgeon, stabilising after 15 limbs.ConclusionELA of the incompetent GSV is effective and safe. ELA is simple to perform, well accepted by patients and relatively atraumatic. In our opinion, ELA can be easily implemented in surgical practice.     

Contaminants from the transplant contribute to intimal hyperplasia associated with microvascular endothelial cell seeding.

OBJECTIVES: seeding prosthetic grafts with fat-derived microvascular endothelial cells (MVEC) results not only in a non-thrombogenic EC layer, but also in intimal hyperplasia. Here we investigated incidence, composition, progression, and cause of this intimal hyperplasia. DESIGN: EPTFE grafts with MVEC were implanted as carotid interpositions in six dogs with 1 month, and in three dogs with 4, 8 and 12 months follow-up. Grafts seeded without cells, implanted in the contralateral carotid, served as a control. In another three dogs labelled cells were seeded to investigate the contribution of the seeded cells (2-3 weeks). MATERIALS and METHODS: MVEC were isolated from the falciform ligament. Cells were pressure seeded on ePTFE grafts. Labelling was performed using retroviral gene transduction. The grafts were analysed with immunohistochemical techniques. RESULTS: after 1 month, all patent non-seeded grafts (5/6) showed fibrin and platelet deposition, and all patent seeded grafts (5/6) were covered with a confluent endothelial monolayer on top of a multilayer of myofibroblasts, elastin and collagen. After long term follow-up, all non-seeded grafts were occluded, all patent seeded grafts (4 and 12 months) were covered with an EC-layer with intimal hyperplasia underneath. The thickness of the intima did not progress after 1 month. Transduced cells were found in the endothelial monolayer, hyperplastic intima and luminal part of the prosthesis. CONCLUSIONS: MVEC seeding in dogs results in intimal hyperplasia in all patent grafts, which contains myofibroblasts. Contaminants from the transplant contribute to this intimal hyperplasia.     

Endovenous laser therapy: a new treatment for varicose veins

Varicose veins are very common. For years the most commonly applied treatment for great saphenous vein (GSV) insufficiency was saphenofemoral junction ligation with saphenous vein stripping. Minimally invasive methods, such as the endovenous laser therapy, are increasingly used during the last few years. In endovenous laser therapy, a diode laser fibre is inserted percutaneously into the GSV using ultrasonography to confirm the position. Thermal laser energy is applied to the endothelium ofthe GSV, resulting in local venous occlusion. The procedure has rapidly become popular with clinicians who treat varicose veins due to its relative simplicity and high rate of patient satisfaction. Efficacy outcomes are good with an occlusion rate of up to 100%. Recanalisation is rarely occurring even after several years. Pain, haematoma and phlebitis are common adverse events associated with endovenous laser therapy but in most cases are self-limiting. Serious adverse events, such as deep vein thrombosis, are uncommon. The advantages ofendovenous laser therapy are the lack of surgical wounds, so infection and scarring are avoided, and that the procedure can be performed in an outpatient setting using local anaesthesia. Endovenous laser therapy appears to be a safe and effective treatment option for refluxing varicose veins.     

Thrombogenesis of different cell types seeded on vascular grafts and studied under blood-flow conditions

Background: Small-diameter vascular grafts tend to have an early and high occlusion rate. Cell seeding on the luminal surfaces of small-diameter vascular prostheses may provide an antithrombotic lining and improve both the short-term and the long-term patency rates. We studied the net results of procoagulant and anticoagulant properties of seeded grafts under blood-flow conditions, and we compared the different available types of donor cells. Methods: Monolayers of liposuction-derived cultured human microvascular endothelial cells (MVECs), human adult endothelial cells (HAECs), human umbilical vein endothelial cells (HUVECs), and human mesothelial cells (MCs) that had been seeded on expanded polytetrafluoroethylene (ePTFE) grafts were perfused with marginally anticoagulated blood (20 U/mL low molecular weight heparin; shear rate, 400/s, 10 minutes) or with non-anticoagulated blood (shear rate, 100/s, 5 minutes). The thrombin and fibrin generation in time was studied with the measurement of the plasma levels of prothrombin fragment 1 and 2 (F 1+2) and of fibrinopeptide A (FPA). The plain ePTFE graft was taken as a control. Results: When the seeded MCs were perfused with recirculating anticoagulated blood, a linear generation of F 1+2 in time was seen, with high levels of F 1+2 and FPA after 10 minutes (4.38 nmol/L and 362 ng/mL, respectively). Allopurinol was added, and the MCs generated less F 1+2 than the HAECs (0.7 nmol/L vs 1.86 nmol/L; P < .05). No fibrin formation was seen. The MVECs generated low amounts of F 1+2 (0.7 nmol/L; 10 minutes), and the HUVECs and the plain ePTFE graft generated the lowest amounts of F 1+2 (0.26 and 0.25 nmol/L, respectively). When the MCs were perfused with non-anticoagulated blood, high amounts of thrombin and fibrin were generated immediately and constantly and could not be decreased with allopurinol. The perfusion of the plain ePTFE graft showed a dramatic increase in F 1+2 and FPA levels towards the end of the experiments. The seeded HAECs, HUVECs, and MVECs inhibited this increase. These results were confirmed by means of scanning electron microscopy. Conclusion: Vascular prostheses that are seeded with cultured MCs are highly procoagulant. Standard ePTFE graft prostheses also initiate coagulation, which supports the idea of cell seeding. The endothelial cells, of which the MVECs are the most readily available, seem to preserve their anticoagulant properties after being seeded on vascular grafts. (J Vasc Surg 1998;28:1094-1103.)     

Reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and intimal hyperplasia.

INTRODUCTION: fat derived microvascular endothelial cells (MVEC) seeded on prosthetic vascular grafts, improve patency in animals. Results in humans were disappointing, due to thrombogenicity and progressive intimal hyperplasia. Also in animals intimal hyperplasia was found. We postulate that contaminating cells present in the transplant are involved in the intimal hyperplasia. We developed a method to further purify human MVEC from 40-90%. Here we tested the effects of enrichment upon thrombogenicity and seeding-related intimal hyperplasia. METHODS: liposuction fat was enzymatically digested and centrifuged. To enrich MVEC, contaminating macrophages and fibroblasts were removed with dynabeads coated with macrophage- and fibroblast-specific antibodies. Thrombogenicity was assessed by measuring tissue factor and thrombomodulin activity, presence of endothelial nitric oxide synthase and via perfusion of the cells with whole blood. To investigate seeding-related intimal hyperplasia, PTFE grafts were seeded with the cells and cultured for 3 weeks. RESULTS: tissue factor activity of purified cells was reduced compared to nonpurified cells. Purified cells showed thrombomodulin activity and eNOS expression. Fragment 1+2 and Fibrinopeptide A generation after perfusion of purified cells were significantly lower than after perfusion of nonpurified cells, and only nonpurified cells were covered with platelets and fibrin. Prostheses seeded with nonpurified cells showed an EC monolayer above a multilayer of myofibroblasts, prostheses seeded with purified cells only showed a single EC monolayer. Mixing experiments with human umbilical cord EC (HUVEC) and fibroblasts showed that when more than 25% HUVEC were present a confluent EC layer was formed. When the amount of fibroblasts was 25% or less, no development of a subendothelial multilayer of myofibroblasts was found within 3 weeks. CONCLUSION: reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and might prevent seeding-related intimal hyperplasia.