Sandro Lepidi

Shear stress induces changes in the morphology and cytoskeleton organisation of arterial endothelial cells

OBJECTIVES The aim of this study was to determine the changes in the morphology and cytoskeleton organisation of endothelial cells (EC) determined by exposure to a laminar flow. Cultured EC were exposed to a wall shear stress of 6 dyne/cm2 for 24 hours. CHIEF OUTCOME MEASURES The morphology of EC was analysed by light and scanning electron microscopy. The organisation of the cytoskeleton was determined by double fluorescence labeling with antibody anti-vimentin, anti-vinculin, anti-tubulin, and with rhodamine-labeled phalloidin. RESULTS EC exposed to laminar flow become round-shaped with decreased area of adhesion to the substrate. There was a clear reorganisation of the cytoskeleton after exposure to shear stress; the distribution of actin changed from a stress fibre pattern to a more diffuse membrane-associated distribution. These changes in shape and cytoskeleton organisation were reversible after a 48-hour resting period. CONCLUSIONS EC respond to laminar flow in a predictable manner and these findings may be correlated to the functional changes of EC observed after exposure to shear stress.

Shear Stress Influences the Release of Platelet Derived Growth Factor and Basic Fibroblast Growth Factor by Arterial Smooth Muscle Cells

OBJECTIVES To determine the correlation between haemodynamic forces and the release of two mitogens for smooth muscle cells (SMC): Platelet Derived Growth Factor (PDGF) and basic Fibroblast Growth Factor (bFGF). METHODOLOGY Bovine aortic smooth muscle cells were seeded on fibronectin coated polystyrene cylinders and allowed to reach confluence. The cells were subjected to a laminar flow of 50 cc/min (3 dyne/cm2), 100 cc/min (6 dyne/cm2) and 150 cc/min (9 dyne/cm2) in an in vitro system. Control cells were subjected to similar incubation conditions without flow. PRINCIPAL RESULTS Shear stress increased the release of mitogens by SMC. The release of mitogens was proportional to the level of shear stress and was still evident 24 hours after flow cessation. Conditioned serum-free medium from SMC subjected to shear stress increased tritiated thymidine uptake in Swiss 3T3 fibroblasts 13-fold as compared to conditioned serum-free medium from control SMC not subjected to shear stress (p < 0.01) and threefold as compared to standard control (p < 0.001). Addition of an excess of anti-PDGF antibody reduced the mitogenic activity of the conditioned medium by 30% (p < 0.01). Addition of an excess of anti-bFGF antibody reduced the mitogenic activity of the conditioned medium by 60% (p < 0.01). CONCLUSIONS Increasing shear stress promotes the release of both PDGF and bFGF from arterial SMC in culture and is a possible explanation for atherosclerosis formation.

Formation of myointimal hyperplasia and cytokine production in experimental vein grafts.

BACKGROUND The purpose of this study was to determine the correlation between progression and regression of myointimal hyperplasia (MH) and cytokine production in experimental vein grafts. Although the autologous vein is the best suitable bypass conduit for reconstruction of peripheral arteries, at the end of the first year thrombosis in the coronary and lower extremity circulation ranges from 20% to 50%. Many of these failures are caused by MH. METHODS In 76 inbred Lewis rats, a 1 cm long segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngeneic Lewis rats. In 56 animals the arterial vein graft was explanted 3 days (n = 10), 7 days (n = 10), 4 weeks (n = 26), and 12 weeks (n = 10) after operation. In 20 animals the vein graft was explanted 4 weeks after being in the arterial system and reimplanted as iliac venovenous bypass in syngeneic Lewis rats. These grafts were explanted 2 weeks (n = 10) and 8 weeks (n = 10) later. Grafts were analyzed by light and electron microscopy, morphometric study, and histochemical analysis and were put in an organ culture to assess cytokine production. RESULTS We observed MH formation in arterial vein grafts and MH regression in reimplanted vein grafts (p < 0.001). MH formation was correlated with production of platelet-derived growth factor, basic fibroblast growth factor, interleukin-1, and tumor necrosis factor-alpha. MH regression was correlated with transforming growth factor-beta 1 production. CONCLUSIONS On the basis of the results of our study, we conclude that MH formation in experimental vein grafts depends on production of platelet-derived growth factor, basic fibroblast growth factor, interleukin-1, and tumor necrosis factor-alpha, and MH regression depends on transforming growth factor-beta 1 production. Cytokine therapy may represent a valuable new treatment to prevent vein bypass failures caused by MH.

Progression and regression of myointimal hyperplasia in experimental vein grafts depends on platelet-derived growth factor and basic fibroblastic growth factor production

PURPOSE The factors that lead to myointimal hyperplasia (MH) in arterial vein grafts (AVGs) are unknown. Platelet-derived growth factor (PDGF) and basic fibroblastic growth factor (bFGF) are two powerful mitogens for smooth muscle cells that have been implicated in the genesis of MH. The aim of this study was to analyze the correlation between progression and regression of MH and production of PDGF and bFGF in experimental vein grafts. MATERIALS In 64 inbred Lewis rats, a 1-cm segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngenic rats. In 48 rats, the AVG was explanted 3 days (n = 8), 7 days (n = 8), 4 weeks (n = 24), and 12 weeks (n = 8) after surgery. In 16 rats the vein graft was explanted after being in the arterial system for 4 weeks and was reimplanted as a venous-venous bypass in syngenic Lewis rats. Reimplanted vein grafts (RVGs) were explanted 2 weeks (n = 8) and 8 weeks (n = 8) later. Grafts were analyzed by light and electron microscopy, morphometry, and histochemistry, and were put in organ culture to assess PDGF and bFGF production and mitogenic activity. RESULTS We observed MH formation in AVGs and MH regression in RVGs (p < 0.001).PDGF and bFGF production correlated with the degree of MH (p < 0.01). Histochemistry showed PDGF and bFGF in the area of MH in AVG, which disappeared in RVG. Conditioned media from AVG had greater mitogenic activity than RVG or control veins. CONCLUSION MH formation and regression in experimental vein grafts correlate with PDGF and bFGF production.

Outcomes of endovascular aneurysm repair with contemporary volume-dependent sac embolization in patients at risk for type II endoleak

OBJECTIVE The aim of this study was to evaluate outcomes of intraoperative aneurysm sac embolization during endovascular aneurysm repair (EVAR) in patients considered at risk for type II endoleak (EII), using a sac volume-dependent dose of fibrin glue and coils. METHODS Between January 2012 and December 2014, 126 patients underwent EVAR. Based on preoperative computed tomography evaluation of anatomic criteria, 107 patients (85%) were defined as at risk for EII and assigned to randomization for standard EVAR (group A; n = 55, 44%) or EVAR with intraoperative sac embolization (group B; n = 52, 42%); the remaining 19 patients (15%) were defined as at low risk for EII and excluded from the randomization (group C). Computed tomography scans were evaluated with OsiriX Pro 4.0 software to obtain aneurysm sac volume. Freedom from EII, freedom from EII-related reintervention, and aneurysm sac volume shrinkage at 6, 12, and 24 months were compared by Kaplan-Meier estimates. Patients in group C underwent the same follow-up protocol as groups A and B. RESULTS Patient characteristics, Society for Vascular Surgery comorbidity scores (0.99 ± 0.50 vs 0.95 ± 0.55; P = .70), and operative time (149 ± 50 minutes vs 157 ± 39 minutes; P = .63) were similar for groups A and B. Freedom from EII was significantly lower for group A compared with group B at 3 months (58% vs 80%; P = .002), 6 months (68% vs 85%; P = .04), and 12 months (70% vs 87%; P = .04) but not statistically significant at 24 months (85% vs 87%; P = .57). Freedom from EII-related reintervention at 24 months was significantly lower for group A compared with group B (82% vs 96%; P = .04). Patients in group B showed a significantly overall mean difference in aneurysm sac volume shrinkage compared with group A at 6 months (-11 ± 17 cm(3) vs -2 ± 14 cm(3); P < .01), 12 months (-18 ± 26 cm(3) vs -3 ± 32 cm(3); P = .02), and 24 months (-27 ± 25 cm(3) vs -5 ± 26 cm(3); P < .01). Patients in group C had the lowest EII rate compared with groups A and B (6 months, 5%; 12 months, 6%; 24 months, 0%) and no EII-related reintervention. CONCLUSIONS This randomized study confirms that sac embolization during EVAR, using a sac volume-dependent dose of fibrin glue and coils, is a valid method to significantly reduce EII and its complications during early and midterm follow-up in patients considered at risk. Although further confirmatory studies are needed, the faster aneurysm sac volume shrinkage over time in patients who underwent embolization compared with standard EVAR may be a positive aspect influencing the lower EII rate also during long-term follow-up.

Growth factor production after polytetrafluoroethylene and vein arterial grafting: an experimental study

PURPOSE Occlusion caused by myointimal hyperplasia appears to be the main reason of late failure of polytetrafluoroethylene (PTFE) arterial bypass grafts. Evidence exists that growth factors are involved in the genesis of myointimal hyperplasia. The aim of this study was to assess the release of platelet-derived growth factor (PDGF) and basic fibroblastic growth factor (bFGF) by PTFE arterial grafts. METHODS In 15 inbred Lewis rats a 1 cm long segment of PTFE was interposed at the level of the abdominal aorta. In a control of another 15 Lewis rats in a vein graft was implanted at the level of the abdominal aorta. Animals were killed four weeks after implantation and the tissue was studied in organ culture for release of PDGF AA, PDGF BB, and bFGF. RESULTS PTFE grafts released a greater quantity of PDGF AA than did control vein grafts (28 +/- 4 ng/cm2/72 hr vs 7 +/- 2 ng/cm2/72 hr). Similarly, PTFE grafts released a greater quantity of bFGF than did arterial vein grafts (308 +/- 22 ng/cm(2)/72hr vs 204 +/- 20 ng/cm2/72 hr). CONCLUSIONS We conclude that PTFE arterial grafts released a high quantity of growth factor, which could explain, in part, the occurrence of distal anastomotic myointimal hyperplasia.

The degree of porosity influences the release of growth factors by healing polytetrafluoroethylene (PTFE) grafts

OBJECTIVES The aim of this study was to determine the influence of the degree of porosity on the release of growth factors (PDGF AA, PDGF BB, bFGF) by healing PTFE grafts. DESIGN AND SETTING Laboratory animal study. MATERIALS 1 cm long segments of non-reinforced PTFE grafts (30 microns fibril length, 2 mm internal diameter, 0.39 mm thick) were placed as an abdominal aortic interposition in Lewis rats. Fifteen grafts served as control (Group A; porous grafts); in eight rats (Group B; non porous grafts) the PTFE graft was completely wrapped by a non-porous plastic envelope. Animals were killed 4 weeks after surgery. OUTCOME MEASURES The release of PDGF AA, PDGF BB and bFGF was assessed by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS The release of PDGF AA, PDGF BB and bFGF was statistically higher in porous grafts. The only histological difference between the two groups was that porous PTFE grafts were invaded by many tufts of capillaries from the surrounding tissue, whereas this phenomenon was absent in non porous PTFE grafts. CONCLUSIONS The degree of porosity influences the release of growth factors by healing PTFE grafts. This fact may have implication in the endothelisation of PTFE grafts and in myointimal hyperplasia formation as well.

bFGF release is dependent on flow conditions in experimental vein grafts

OBJECTIVES Basic Fibroblastic Growth Factor (bFGF) is a powerful mitogen for smooth muscle cells and has been implicated in the genesis of Myointimal hyperplasia. The aim of this study was to determine the release of bFGF by veins in different haemodynamic conditions. DESIGN AND SETTING Laboratory animal study. MATERIALS In 39 Lewis rats, a 1 cm long segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngenic Lewis rats. Arterial Vein Grafts (AVG) were harvested after 4 weeks (AVG 4) and 12 weeks (AVG 12). In 16 animals the arterial vein grafts were explanted 4 weeks after the initial operation and reimplanted (Reimplanted Vein Grafts: RVG) in syngenic Lewis rats as venous-venous bypass grafts at the level of the left iliac vein and harvested after 2 weeks (RVG 2) and 8 weeks (AVG 8). OUTCOME MEASURES The tissue was studied in organ culture in a serum-free system for (1) release of bFGF (immunoassay) and (2) mitogenic activity of the conditioned media. Scanning electron and light microscopy studies were also performed. RESULTS bFGF release by veins increased significantly (p < 0.01) when veins were inserted in the arterial circulation, and decreased significantly (p < 0.01) when grafts were reimplanted in the venous system. bFGF release (ng/cm2): [Formula: see text] CONCLUSION Vein inserted in the arterial circulation release a higher quantity of bFGF. This could explain in part, the formation of myointimal hyperplasia in arterial vein graft.

Commentary on “Efficient Differentiation of Bone Marrow Mesenchymal Stem Cells into Endothelial Cells in vitro ”

Chengen et al. have published an interesting experimental study entitled “Efficient Differentiation of Bone Marrow Mesenchymal Stem Cells into Endothelial Cells in vitro”. They describe an improved method of differentiating human bone marrow mesenchymal stem cells (MSCs) into endothelial cells (ECs). Bone marrow cells from human donors were retrieved; MSCs were isolated by density gradient centrifugation and cell culture in Iscove’s Modified Dulbecco’s Medium supplemented with 10% fetal bovine serum. Adherent cells were grown in the same fresh medium until they displayed a marked spindle shape and the CD marker profile identified the cells as MSCs. A cocktail of growth factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, insulin-like growth factor, and epidermal growth factor was then added with ascorbic acid and heparin, to stimulateMSCs to differentiate into ECs.With this inducingmedium, Chengen et al.were able to generate a larger proportion of ECs than in previous studies. They defined ECs by expression of CD31 and CD34 during immunostaining, and by von Willebrand factor, vascular endothelial cadherin and VEGF receptor 2 in quantitative reverse transcription polymerase chain reaction. They also show that the differentiated ECs were able to grow into tube like structures in Matrigel. The authors report a successful differentiation of up to 60% of MSCs into ECs after 14 days and conclude that these results provide a method to efficiently promote differentiation of MSCs into ECs in vitro for potential application in peripheral arterial disease (PAD). In the last decade, cell based therapies have been explored as a treatment option for patients with PAD with critical limb ischaemia (CLI) with no surgical or endovascular option for revascularisation. Several clinical studies have been conducted to test the efficacy of autologous bone marrow derived cell therapies for the treatment of CLI, ranging from case reports, small series, uncontrolled trials, and randomized controlled trials. The Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation