Elevated platelet-derived growth factor production by aortic grafts implanted on a long-term basis in a canine model.

An endothelial cell lining in a prosthetic vascular graft has been shown to decrease graft thrombogenicity. However, previous studies in our laboratory demonstrated that grafts seeded with endothelial cells produced platelet-derived growth factor, a potent smooth muscle cell mitogen that may promote intimal hyperplasia. This study was undertaken to assess temporal changes in platelet-derived growth factor production by grafts seeded with endothelial cells and unseeded grafts and adjacent arteries. Adult beagles underwent placement of 20 to 22 cm long, 8 mm inner diameter, expanded polytetrafluoroethylene thoracoabdominal aortic grafts that were either seeded with autologous jugular vein endothelial cells or were unseeded controls. Grafts and adjacent arteries were removed at times up to 2 years after implantation. The tissue was studied in organ culture and platelet-derived growth factor production was measured after 72 hours with use of a radioreceptor assay. Platelet-derived growth factor production by endothelialized grafts increased significantly over the period studied, especially at the anastomoses, whereas that by arterial segments did not change significantly. The increase in platelet-derived growth factor production was greater in the distal than the proximal anastomotic segment suggesting a possible explanation for the clinical finding of more severe intimal hyperplasia at the distal anastomosis.     

Prostacyclin production from seeded prosthetic vascular grafts.

Endothelial cell seeding has been proposed as a method of improving patency rates in small-calibre prosthetic vascular grafts. In vivo, endothelial cells normally produce prostacyclin (PGI2), a potent antiplatelet agent. The aim of this study was to determine whether seeded grafts show significant PGI2 production after in vivo implantation. Grafts were seeded with either autologous canine venous endothelial cells or autologous microvascular endothelial cells. After 12 weeks, PGI2 production was assessed under basal and stimulated conditions. Seeded grafts were compared with non-seeded controls and the corresponding aorta. The overall patency rate in seeded grafts was 80 per cent compared with 10 per cent in non-seeded grafts (P < 0.01). Grafts seeded with cells from either source produced significantly more PGI2 than unseeded grafts in both basal and stimulated states (P < 0.05). The aorta produced significantly more PGI2 than seeded grafts under both conditions (P < 0.01). Endothelial cell seeding produces a functional graft and leads to an improved patency rate.     

Enhanced patency of small-diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells

Fourteen adult foxhounds underwent bilateral iliofemoral bypasses with externally supported, knitted Dacron grafts measured 4 mm in internal diameter and 10 cm in length. These conduits were preclotted with 10 ml of blood mixed with 0.5 ml of culture medium. Autologous endothelial cells, enzymatically derived from external jugular veins, were added to blood and medium used to preclot one graft in each dog. The other, unseeded graft served as a control. Grafts were anastomosed, end to end, to the iliac and femoral arteries. All dogs received dipyridamole, 50 mg twice a day for 4 days preoperatively, and aspirin, 5 grains four times a day for 1 day preoperatively. Both drugs were continued 14 days after operation. Grafts were removed from three dogs at 2 and 4 weeks and from four dogs at 8 and 16 weeks. All grafts were patent at 2 weeks during drug administration. Cumulative patency rates beyond 2 weeks were 73% in 11 seeded grafts and 27% in 11 control grafts, a statistically significant difference (P = 0.03). Seeded grafts were completely surfaced with a monolayer of endothelium between 2 and 4 weeks. Small-graft patency appeared related to evolution of endothelial surfaces, the development of which was clearly facilitated by seeding with autologous endothelium.     

Platelet-derived endothelial cell growth factor gene therapy for limb ischemia

OBJECTIVES: Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to thymidine phosphorylase (TP), and it can induce angiogenesis, including arteriogenesis, in chronically ischemic canine myocardium. Because its effect on peripheral arterial disease has not been elucidated, we investigated whether overexpression of PD-ECGF/TP could ameliorate chronic limb ischemia in rabbits. METHODS: Left femoral arteries were resected from 24 male rabbits. After 10 days, a plasmid vector containing human PD-ECGF/TP complimentary DNA was injected into 10 sites in the adductor muscles. Control groups received either the LacZ plasmid vector or saline vehicle only (n = 8 per group). Blood pressure was measured in the calf before surgery, at the onset of ischemia, 10 days later, and 20 and 30 days after gene transfer. Collateral vessel development and limb perfusion were assessed by angiography, and resected tissues underwent molecular and histologic examination. RESULTS: In the PD-ECGF/TP group, human PD-ECGF/TP messenger RNA and protein were still detected at 30 days after treatment. Calf blood pressure decreased significantly after femoral artery resection in all three groups. It subsequently showed a greater increase in the PD-ECGF/TP group than in either control group, and the difference was significant at 20 days after treatment (PD-ECGF/TP, 97.4 +/- 7.4; LacZ, 58.6 +/- 6.9; saline, 41.3 +/- 3.6). Immunohistochemical staining demonstrated an increased ratio of capillaries and arterioles to muscle fibers in the PD-ECGF/TP group (2.14 +/- 0.13 and 1.51 +/- 0.06), but not in the LacZ group (1.39 +/- 0.04 and 0.71 +/- 0.05) or the saline group (1.34 +/- 0.05 and 0.71 +/- 0.04, P < .01). The angiographic score was higher in the PD-ECGF/TP group (0.96 +/- 0.08) than in the LacZ group (0.50 +/- 0.02) or saline group (0.51 +/- 0.03) at 30 days after gene transfer (P < .01). CONCLUSIONS: This study demonstrated that PD-ECGF/TP gene transfer induced angiogenesis and decreased ischemia in a rabbit hindlimb model by promoting arteriogenesis, suggesting that targeting this gene may be a promising therapeutic strategy for peripheral vascular disease.     

Platelet-derived growth factor in Dupuytren's disease.

This study investigated whether platelet-derived growth factor, a potent inducer of cell proliferation, was identifiable in association with myofibroblasts in Dupuytren's disease. Myofibroblasts in the hypercellular disease stages showed a strong reaction to platelet-derived growth factor antibody using light and electron microscopic immunochemical labels. Platelet-derived growth factor may play a role as a cellular signal for myofibroblast proliferation in the formation of the pathognomonic nodule in Dupuytren's disease.     

Are seeded endothelial cells the origin of neointima on prosthetic vascular grafts?

Although endothelial cell seeding has been demonstrated to be effective in producing uniform endothelial coverage on prosthetic grafts, no one has demonstrated that the seeded cells are the genetic precursors of the endothelial cells that ultimately line the graft. This study was performed to determine whether seeded endothelial cells were the source of the neoendothelial surface on Dacron vascular grafts implanted in the pig. Eight pairs of littermate male and female pigs were selected at birth and allowed to grow until their weight reached 20 to 25 kg. Endothelial cells were enzymatically harvested from the jugular vein of the female siblings and seeded onto 8 mm diameter grafts, 25 cm in length. The grafts were then implanted as a thoracoabdominal bypass into the male siblings. Seven other randomly selected pigs of similar size (four males and three females) had thoracoabdominal bypass with similar grafts, which were preclotted with autologous blood only, but not seeded. After 4 weeks, grafts in both groups were covered with a neointima that was more than 90% thrombus-free. The graft surface cells were grown in culture and were documented to be endothelium. Chromosome analysis demonstrated that the surface cells on the seeded grafts were from the male host and did not originate from the donor female cells. We conclude that seeded endothelial cells are not the source for the neointima on Dacron grafts in the porcine model. In addition, we have again documented that spontaneous endothelial coverage of grafts does occur without endothelial seeding.     

Platelet-derived growth factor and vascular endothelial growth factor expression in disc herniation tissue: an immunohistochemical study

Angiogenesis is essential in tissue growth and regeneration. There are several factors that are able to stimulate vascular endothelial cell growth, including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Disc herniation tissue (DHT) contains vascular ingrowth, which promotes granulation tissue formation. In this study we observed 50 disc herniations for PDGF and VEGF immunoreactivity. PDGF immunopositivity was detected in 38 samples (78%). In 28 samples (56%) there were PDGF immunopositive capillaries, PDGF immunopositive disc cells were detected in 19 samples (38%) and PDGF immunopositive fibroblasts in 6 DHT samples (12%). VEGF immunopositive capillaries were identified in 44 DHT samples (88%). For neither growth factor was immunopositivity dependent on preoperative radicular pain duration. In extrusions (n = 25) VEGF immunopositive capillaries were detected in 23 samples (92%) and PDGF immunopositivity in 21 samples (84%). PDGF immunopositivity was more commonly associated with capillaries than with nuclei of disc cells. In sequesters (n = 20) VEGF immunopositive capillaries were identified in all samples and PDGF immunopositivity in 16 (80%). As in extrusions, PDGF immunoreaction was more prevalent in capillaries than in disc cells. Patient age did not relate to VEGF expression. In all age groups it was higher than 80%. Thus capillaries in disc herniation tissue are evidently newly formed and our results demonstrate that PDGF and VEGF participate in the neovascularization process. The presence of PDGF in fibroblasts and in disc cells suggests that this growth factor regulates the function of these cells, possibly the proliferation of the cells and the production of extracellular matrix components.     

Improved retention of endothelial cells seeded on polyurethane small-diameter vascular grafts modified by a recombinant RGD-containing protein

Sponge-type small-diameter vascular grafts were fabricated from a medical-grade polyurethane, Pellethane 2363-80A, by utilization of a salt casting technique. The grafts were compliance matched with a storage modulus of 0.53 +/- 0.08 MPa. The luminal surface of grafts was modified with a thin layer ( approximately 40 micro m) of gelatin crosslinked by epoxide. Then a special Arg-Gly-Asp (RGD)-containing recombinant protein, named CBD-RGD (cellulose binding domain RGD-containing protein), was coated onto the gelatin layer. The platelet adhesion and activation on such a gelatin/CBD-RGD modified surface was significantly reduced. Human umbilical vein endothelial cells were seeded more efficiently onto the modified grafts. There was also a substantial reduction in the subsequent loss of cells from the graft surface following perfusion in vitro. The cell number retained on the modified graft was enhanced by three times after 1 h of perfusion, and by eight times after 3 h of perfusion (retention rate approximately 63%). The retention after 3 h of perfusion could be further increased to nearly 100% if the lined endothelium on gelatin/CBD-RGD modified graft was cultured for another week before perfusion. The modified surface was also shown to help canine external jugular vein endothelial cells to maintain the round cell morphology in vitro.     

Vascular endothelial growth factor production and regulation in human peritoneal mesothelial cells

BACKGROUND: Vascular endothelial growth factor (VEGF) was recently found in peritoneal effluents of peritoneal dialysis (PD) patients. It was suggested that human peritoneal mesothelial cells (HMC) contribute to the intraperitoneal production of VEGF, which may augment vascular permeability, vasodilation and neoangiogenesis in the peritoneal membrane. The present study was designed to assess the influence of proinflammatory cytokines, thrombin, d-glucose and glycated albumin in the regulation of VEGF synthesis in primary HMC cultures. METHODS: VEGF antigen concentrations were measured in the cell supernatant by ELISA and VEGF mRNA expression was evaluated by real time RT-PCR. RESULTS: Incubation of HMC with interleukin-1alpha (IL-1alpha; 10 to 100 U/mL), tumor necrosis factor-alpha (TNF-alpha; 500 to 1000 U/mL) or thrombin (1 to 10 U/mL) resulted in a time (24 to 72 hours) and concentration dependent increase in VEGF synthesis. In contrast, d-glucose (30 to 90 mmol/L), which is commonly used as an osmotic agent in peritoneal dialysis, was not able to up-regulate VEGF expression. High glucose levels even decreased VEGF production. However, exposure of HMC to Amadori-modified glycated albumin, which is generated in the peritoneal cavity in the presence of glucose-based dialysis solutions, resulted in a dose and time dependent increase in VEGF mRNA expression and antigen secretion. CONCLUSIONS: These results demonstrate, to our knowledge for the first time, that glycated serum albumin, not glucose, increases VEGF production in HMC. HMC play an important role as a source of intraperitoneal VEGF synthesis, and VEGF expression also is up-regulated in the presence of proinflammatory cytokines and thrombin. Additionally, these results confirm clinical data that the continuous exposure of the peritoneal membrane to glucose-based dialysis solutions is an important stimulus for VEGF expression. However, it is not glucose per se, but nonenzymatic glycation products like glycated albumin that up-regulate VEGF expression.     

子宫内膜异位症腹腔巨噬细胞释放血管内皮细胞生长因子能力的变化

目的 :探讨子宫内膜异位症腹腔巨噬细胞释放血管内皮细胞生长因子( VEGF)的变化。方法 :将行诊断性腹腔镜检查的不育患者 45例 ,根据术后诊断分为子宫内膜异位症组 ( 2 5例 )和对照组 ( 2 0例 ) ,采用酶联免疫吸附测定检测腹腔液和腹腔巨噬细胞培养上清液中 VEGF的水平。结果 :子宫内膜异位症患者腹腔液及腹腔巨噬细胞培养上清液中 VEGF的水平均显著高于对照组 ( P<0 .0 1 )。结论 :腹腔巨噬细胞分泌能力增强可能是导致子宫内膜异位症患者腹腔液 VEGF水平增高的原因     

Platelet-derived growth factor in experimental glomerulonephritis

Growth factors and in particular platelet-derived growth factor(PDGF) have been implicated in the pathogenesis of glomerulonephritisand glomerulosclerosis. We have studied the distribution ofimmunoreactive PDGF (iPDGF) within serial kidney biopsies (days7, 15, 30, 90 and 120) of eight rats with an accelerated formof nephrotoxic serum nephritis (NTN). The course of NTN wasmild in five rats and severe in three. Two patterns of immunostainfor PDGF were noted. The first consisted of iPDGF-B chain ina glomerular segmental distribution similar to that of infiltratingmonocytes (OX6+cells). At all stages of NTN the distributionof iPDGF-B chain correlated closely with the immunostain formonocytes. The second pattern of immunostain showed iPDGF-Achain in a diffuse distribution along the glomerular capillarylining and to a lesser extent in some mesangial cells. In severelyaffected rats the magnitude of the iPDGF-A increased along withglomerulosclerosis but disappeared later from areas of segmentaland global glomerular obsolescence. By contrast, in rats withmilder NTN glomerular iPDGF-A chain peaked early and decreasedsubsequently. During the acute phase of NTN, on day 7, iPDGFA chain correlated with the severity of proteinuria (r=0.848,P<0.05) and that of glomerular cellular proliferation (r=0.91,P<0.02). On day 30 iPDGF-B correlated positively with proteinuria(r=0.998, P<0.01, n=5) and with serum creatinine (r=0.949,P<0.02, n=5). During the late stages of NTN, both iPDGF-A(day 90, r=0.884, P<0.05) and iPDGF-B (day 120, r=0.941,P<0.01) correlated closely with the extent of glomerulosclerosis.Both PDGF chains (A and B) are detectable through out the courseof nephrotoxic nephritis in rats. PDGF may play a role in theearly proliferative and late sclerotic changes of experimentalglomerulonephritis.     

Growth factor production by polytetrafluoroethylene vascular grafts

In earlier studies, we have shown that porous (60 micron internodal distance) PTFE grafts develop a complete endothelial layer 2 weeks after being implanted in baboons. Subsequently, the intima of the graft thickens on account of SMC proliferation only where an overlying endothelial layer is present. SMCs in normal endothelialized artery proximal and distal to the graft show no detectable proliferation. The purpose of this study was to investigate the possibility that growth factors released from the graft endothelium or SMCs regulate SMC proliferation. PTFE grafts (4 mm I.D., 60 micron internodal distance) were placed in the aortoiliac circulation of baboons and removed at 2 weeks. The grafts were perfused ex vivo with tissue culture medium (Ham's F12 + 25 mmol/L HEPES and 2% calf plasma-derived serum) at 2.5 ml/hr for 5 hours. Perfused native carotid, aorta, and femoral arteries served as controls. After this period of perfusion, graft and arterial endothelium was intact as shown by scanning electron microscopy. The mitogenic activity (thymidine incorporation) of the perfusates was measured in an assay with quiescent 3T3 cells and baboon aortic SMCs and corrected for the surface area of the perfused vessels. These studies demonstrated markedly increased mitogenic activity in the perfusates of grafts compared with perfusates of native vessels. These results provide support for the hypothesis that the vascular wall cells in healing grafts can produce factors that regulate smooth muscle cell growth.     

Platelet-derived growth factor, basic fibroblast growth factor, and interferon gamma increase type IV collagen production in human fetal mesangial cells via a transforming growth factor-beta-dependent mechanism.

BACKGROUND: Glomerulosclerosis is characterized by glomerular accumulation of extracellular matrix following mesangial cell proliferation. The precise pathomechanism of glomerulosclerosis is still undetermined. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (b-FGF) are known to be mitogenic for mesangial cells, and interferon gamma (IFN-gamma) is known to have an inhibitory effect on mesangial cell proliferation. We attempted to clarify the role of these cytokines on mesangial matrix production using cultured human fetal mesangial cells (HMC). METHODS: HMC were incubated with these cytokines for 24-72 h and the levels of type IV collagen and TGF-beta in the cell supernatants were measured by enzyme immunoassay. RESULTS: PDGF, b-FGF, and IFN-gamma stimulated type IV collagen production by HMC in a dose- and time-dependent manner. The anti-TGF-beta neutralizing antibody clearly inhibited their stimulatory effect on type IV collagen production. PDGF and b-FGF also stimulated TGF-beta production by HMC in a dose-dependent manner, although IFN-gamma did not. CONCLUSION: PDGF, b-FGF, and IFN-gamma stimulate type IV collagen production in cultured HMC via a TGF-beta-dependent mechanism.     

Precoating substrate and surface configuration determine adherence and spreading of seeded endothelial cells on polytetrafluoroethylene grafts

Primary adherence and attachment area of seeded human endothelial cells (EC) were determined on differently coated polytetrafluoroethylene (PTFE) grafts. Cell counts and morphometric analyses were done immediately after 60 minutes of electronically controlled seeding of 3 x 10(4) EC/cm2, as well as after 3 hours of subsequent incubation. Cell adherence and cell spreading were distinctly superior on two surface-covering substrates: fibronectin-treated type I/III collagen and fibrinolytically inhibited fibrin glue. Uncovered, purely fibronectin- or laminin-coated PTFE or type IV collagen treated with the specifically binding glycoprotein laminin showed a far lower EC attachment rate and less pronounced cell spreading. It appears that not only a high surface content of fibronectin but also a smooth PTFE-covering matrix are prerequisites for optimal primary adherence and cell spreading. Because fibrin glue might be fibrinolytically degraded despite its plasmin-inhibiting epsilon-amino-caproic acid compound, type I/III collagen plus fibronectin could provide an optimal precoating substrate for EC lining of PTFE grafts.     

Vascular endothelial growth factor promotion of neoangiogenesis in conventional nerve grafts

The effect of vascular endothelial growth factor (VEGF) on angiogenesis and neovascularization of conventional nerve grafts was evaluated in 48 rabbits. A 2.5-cm segment of right sciatic nerve was removed and orthotopically repaired. This graft was wrapped in dialysis tubing to prevent vessel ingrowth from adjacent tissue. An osmotic pump delivered either VEGF (100 ng/h for 3 days) or control solution. Evaluation methods included angiography, vessel density, and nerve blood flow measurement at 3, 7, and 14 days. On day 3, 42% of the control nerves and 100% of VEGF-treated nerves had partial longitudinal neovascularization. Vessel density (0.84 +/- 0.22 vessel/mm(2)) and nerve blood flow [25.34 +/- 7.62 mL/(min.100 g)] in VEGF-treated nerves were significantly higher than control group values [0.23 +/- 0.13 vessel/mm(2) and 5.35 +/- 0.99 mL/(min.100 g)]. Progressive improvement in parameters was seen at 7 and 14 days. Vascular endothelial growth factor infusion accelerates longitudinal neoangiogenesis and shortens the nerve ischemic time to 3 days in this model. The revascularization of VEGF-treated conventional nerve grafts in a poorly vascularized bed is identical to that of grafts in a healthy bed.