Graft Smooth Muscle Cells Research Articles

Effect of treatment of jugular vein graft with glutaraldehyde on intimal hyperplasia in rabbits

Objective To investigate the effect of treatment of jugular vein graft with glutaraldehyde on intimal hyperplasia in rabbits. Methods Sixty New Zealand white rabbits were randomly divided into experimental group and the control group. Before the anastomosis, the vein in the experimental group was soaked in 1% glutaraldehyde solution for 10 min, and that in control group in normal saline for 10 min. The vein graft was harvested after 4 and 12 weeks. HE staining, smooth muscle cell α-actin and proliferating cell nuclear antigen ( PCNA ) immunohistochemical staining were performed. Apoptosis was detected by TUNEL. Results Four and 12 weeks after grafting, the number of smooth muscle cells in the experimental group was significantly less than that in the control group, and intima thickness [(0. 016 ±0. 004), (0. 031 ±0. 008) mm]in the experimental group was thinner than in the control group [(0. 025± 0. 005), (0. 093 ± 0. 024 ) mm, ( P 0. 05 ). The apoptosis rate in the experimental group was higher than in the control group [(3. 3 ± 0. 7 ) %, (6. 7 ± 1. 5) % vs ( 1.2 ± 0. 4) %, ( 1.8 ±0. 5 ) %]in 4 and 12 weeks after grafting ( P < 0. 05 ). Conclusion Treatment of vein graft with glutaraldehyde can significantly reduce the number of graft smooth muscle cells and inhibit intimal hyperplasia. Key words: Glutaraldehyde; Vein graft; Intimal hyperplasia; Apoptosis

Synthetic smooth muscle cell phenotype is associated with increased nicotinamide adenine dinucleotide phosphate oxidase activity: Effect on collagen secretion

Smooth muscle cells (SMCs) from prosthetic vascular grafts secrete higher levels of collagen than aortic SMCs under basal conditions and during incubation with oxidized low-density lipoprotein. We postulated that reactive oxygen species (ROS) contributed to the observed difference. The objective of this study was to assess the effect of ROS on collagen secretion by aortic and graft SMCs and explore the mechanism involved. SMCs isolated from canine aorta or Dacron thoracoabdominal grafts were incubated with 6-anilinoquinoline-5,8-quinone (LY83583), an agent that induces superoxide production. Type I collagen in the conditioned medium was measured by enzyme-linked immunosorbent assay, and superoxide anion production was measured by lucigenin assay. LY83583 stimulated a rapid increase in collagen production by graft SMCs that paralleled the LY83583-induced increase in superoxide production. The increase in both collagen and superoxide was greater in graft SMCs than aortic SMCs. Collagen and superoxide production were inhibited by superoxide scavengers. Nicotinamide adenine dinucleotide phosphate (NADPH) induced significantly more superoxide production by graft SMCs than aortic SMCs, suggesting that the NADPH oxidase system was more active in graft SMCs. NADPH oxidase inhibitors blocked the superoxide and collagen production induced by LY83583. In SMCs, the synthetic phenotype is associated with increased NADPH oxidase activity and elevated superoxide production in response to an oxidative stress. Superoxide, in turn, leads to increased collagen production. The inflammatory process after prosthetic vascular graft implantation causes oxidative stress that can stimulate collagen production by graft SMCs, contributing to the progression of intimal hyperplasia. The exaggerated response of graft SMCs to oxidative stress offers a potential target for therapeutic interventions.

Allogenic immune response promotes the accumulation of host-derived smooth muscle cells in transplant arteriosclerosis

Smooth muscle cells (SMCs) involved in intimal hyperplasia during transplant vasculopathy are derived both from the graft and the host. Here, the role of an allogenic immune response in the accumulation of host-derived SMCs in the neointima was explored. Infrarenal aorta was transplanted from female F344 to male Lewis rats with or without immunosuppression by cyclosporine A (CsA). Accumulation of host-derived SMCs and inflammatory cells in the grafts, SMC proliferation, and apoptosis were analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR) for the SRY gene. Finally, SMCs were seeded in an allogenic or isogenic manner after balloon injury to carotid arteries and SMC survival was estimated. Proliferating graft SMCs and infiltrating leukocytes were observed in the intima early after transplantation. In parallel, inflammatory cells and immunoglobulins infiltrated the media and apoptosis of medial SMCs occurred, leading to destruction of this layer. CsA decreased the number of SRY+ SMCs in the lesions, restricted medial destruction, and improved survival of allogenic SMCs after seeding in injured arteries. Development of intimal thickenings during transplant vasculopathy involves an allogenic immune response, which promotes accumulation of host-derived SMCs and apoptosis of resident graft SMCs.

Differential PDGF secretion by graft and aortic SMC in response to oxidized LDL.

Smooth muscle cells (SMC) from prosthetic vascular grafts constitutively secrete higher levels of platelet-derived growth factor-AA (PDGF-AA) than aortic SMC. Lipid oxidation products accumulate in grafts and may stimulate PDGF production. The effect of oxidized low-density lipoprotein (oxLDL) on PDGF-AA secretion by aortic and graft SMC was compared. SMC isolated from canine thoracic aorta or Dacron thoracoabdominal grafts (n = 10) were incubated with native LDL or oxLDL (0-400 microg/ml) for 72 h. PDGF-AA in the conditioned medium was measured with enzyme-linked immunosorbent assay. OxLDL increased PDGF-AA production by graft SMC from 78 +/- 2 to 256 +/- 16 pg PDGF/microg DNA and aortic SMC from 21 +/- 1 to 40 +/- 2 pg PDGF/microg DNA. Native LDL had no effect. N-acetylcysteine inhibited oxLDL-induced PDGF increase. Both superoxide and H(2)O(2) stimulated PDGF secretion by graft SMC had little effect on aortic SMC. Our results suggest that PDGF production by graft (synthetic) SMC is more sensitive to stimulation by oxidative stress than aortic (contractile) SMC. Lipid oxidation products that accumulate in prosthetic vascular grafts can cause an oxidative stress, which stimulates PDGF production by graft SMC. PDGF can induce migration of aortic SMC onto the graft, contributing to the development of intimal hyperplasia.

Reduction of Vein Graft Disease Using Photodynamic Therapy with Motexafin Lutetium in a Rodent Isograft Model

Background —Motexafin lutetium (Lu-Tex) is a photosensitizer that targets atheromatous plaque. Subsequent photoactivation (photodynamic therapy [PDT]) induces local cytotoxic effects. The aim of the present study was to investigate whether Lu-Tex targets vein graft intimal hyperplasia and whether subsequent photoactivation attenuates the disease process. Methods and Results —The subcellular localization of Lu-Tex and postillumination viability were studied in cultured human vein graft smooth muscle cells. Inferior vena cava–grafted rats were injected with Lu-Tex (10 mg/kg) 4 or 12 weeks after grafting. Biodistribution was assessed in a subgroup 24 hours after administration. Light therapy (742 nm) was performed 24 hours after Lu-Tex injection by illuminating intraperitoneally placed isografts using a laparotomy. Animals were divided into the following 4 groups: PDT (n=15), Lu-Tex injection and laparotomy (n=13), light treatment (n=14), and laparotomy only (n=13). Grafts were harvested 14 days after treatment for histochemical analysis. Lu-Tex localized within subcellular organelles of smooth muscle cells, and subsequent photoactivation induced cell death via apoptosis. The Lu-Tex concentrations present in the vein grafts were 9.3 times higher than those in the normal inferior vena cava. Postoperative PDT at 4 weeks after surgery significantly reduced the intima/media ratio, whereas treatment at 12 weeks did not reduce the intima/media ratio. Activated macrophages were observed 4 weeks after grafting; however, a significant reduction occurred in these cells by 12 weeks. The mechanism by which PDT works may be related to the presence of activated macrophages. Conclusions —PDT significantly reduces the intima/media ratio in the early phase of vein graft disease. Lu-Tex–mediated PDT may be a viable method for the attenuation of atherosclerotic disease in vein grafts.

The proliferative response to platelet-derived growth factor of smooth muscle cells isolated from synthetic vascular grafts in a canine model

Objective: Previous studies on graft healing have shown increased platelet-derived growth factor (PDGF) production in graft segments versus native aortic segments. The purpose of this study was to characterize the proliferative response of graft smooth muscle cells (SMCs) to PDGF. Methods: Thoracoabdominal grafts were implanted in beagles. SMCs were harvested from the graft and the proximal and distal aortas. Basal proliferation was assessed with growth curves in primary culture. The proliferative response to PDGF then was compared with [ 3H]thymidine uptake studies and cell counts. Finally, PDGF receptors were characterized with radio-labeled ligand binding assays. Results: The growth curves showed that the graft SMCs entered log-phase growth 2 days earlier than did the aortic SMCs. Stimulation of quiescent early-passage graft SMCs with PDGF (10 ng/mL) resulted in a 1.7 ± 0.1–fold increase in [ 3H]thymidine incorporation, which was significantly less than that of the SMCs from both the proximal aorta (11.8 ± 3.0) and the distal aorta (10.2 ± 1.9; P < .5). Similarly, the 1.1 ± 0.1–fold increase in graft SMC cell number was significantly less than the increases for both proximal (2.8 ± 0.5) and distal (2.9 ± 0.8) aortic SMCs ( P < .5). Binding studies on quiescent first-passage cells showed fewer PDGF receptors available for binding in the graft SMCs (185 ± 70 fmol/million cells) as compared with both the proximal (419 ± 147 fmol/million cells) and the distal (387 ± 112 fmol/million cells) aortas ( P < .5). Binding affinity was similar for the three groups. Conclusion: Graft SMCs exist in a chronic proliferative state but exhibit a decreased proliferative response to PDGF and have fewer receptors available for binding PDGF than do aortic SMCs in vitro. (J Vasc Surg 1999;29:845-51.)

Changes in the organization of the smooth muscle cells in rat vein grafts.

Mechanical tensile stress in vein grafts increases suddenly under the influence of arterial blood pressure. In this study, we examined the influence of increased tensile stress on the organization of the smooth muscle cells (SMCs) in the neointima and media of the rat vein grafts. An autogenous jugular vein was grafted into the abdominal aorta of the rat, and changes in the organization of the vein graft SMCs were studied by observing the distribution of SMC actin filaments and nuclei at 3 min and 1, 5, 10, and 30 days after surgery. In a normal jugular vein, the average wall circumferential tensile stress was approximately 3 kPa at an internal pressure of 3 mm Hg. The SMCs, that contained long, slender actin filamentous bundles, were oriented mainly in the circumferential direction of the vessel, and constituted a 2- to 3-cell-thick medial layer underneath the endothelium. In a vein graft, the wall circumferential tensile stress suddenly increased by approximately 140 times compared with the control level. In response to this suddenly increased stress, the SMC layer was stretched into a structure with scattered pores and disrupted SMC actin filamentous bundles within 3 min. This initial change was followed by a rapid reduction in the density of the SMC nuclei and actin filaments within 1 day and progressive SMC proliferation, that was associated with medial thickening and a change in the SMC orientation from 5 to 30 days. Further studies showed that a local inflation of normal jugular veins to 120 mm Hg for 3 min induced a similar change as found in the vein grafts, whereas the organization of the SMCs was not significantly changed in vein-vein grafts, that did not experience a change in tensile stress. These results suggested that increased tensile stress contributed to the initial damage of the SMCs and played a role in the regulation of medial SMC remodeling in vein grafts.

Platelet-derived growth factor production by cells from Dacron grafts implanted in a canine model.

Previous studies of grafts implanted in dogs documented a time-dependent increase in platelet-derived growth factor (PDGF) production that correlated with inner-capsule thickness. The purpose of this study was to identify the cells in vascular grafts that produce PDGF. Dacron thoracoabdominal grafts were seeded with autologous endothelial cells (ECs), implanted in 11 beagles, and removed after 4 or 20 weeks. ECs and smooth muscle cells (SMCs) were cultured from grafts and adjacent aorta, and PDGF in the conditioned media was measured by radioreceptor assay. The PDGF A-chain mRNA level in freshly harvested cells was assessed using reverse transcriptase, followed by polymerase chain reaction, and expressed as a ratio of glyceraldehyde-3-phosphate dehydrogenase signal. Localization of PDGF A-chain and B-chain protein was also examined with immunohistochemical analysis. Graft and aortic ECs in primary culture did not produce significantly different amounts of PDGF in 72 hours, averaging 368 +/- 160 and 340 +/- 81 pg/microgram DNA, respectively. Graft SMCs in primary culture produced significantly more PDGF than aortic SMCs (584 +/- 343 and 113 +/- 94 pg/microgram DNA, respectively; p < 0.01). Graft SMC PDGF secretion remained greater than aortic SMC PDGF secretion through at least six cell passages. PDGF A-chain mRNA levels were not significantly different for aortic or graft ECs. The PDGF A-chain mRNA level was significantly higher for graft SMCs than aortic SMCs (2.44 +/- 0.67 and 1.45 +/- 0.57 pg/microgram, respectively; p < 0.03). Immunocytochemical analysis detected PDGF A-chain and B-chain protein in the ECs from both native aorta and graft as well as the subendothelial SMCs in the graft, but not in the SMCs of the native aorta. These results suggest that graft SMCs are functionally altered, producing more PDGF than aortic SMCs. PDGF produced by graft SMCs may contribute to the development of intimal hyperplasia.

1030-13 Alterations in Endothelial Cell Adenosine Receptors Mediate Endothelium-Dependent Vasoconstriction in Experimental Vein Grafts

Adenosine is a potent endogenous vasodilator which mediates its action through A1 and A2 receptor subtypes located on both vascular endothelial and vascular smooth muscle cells (SMC). Adenosine-mediated relaxation in vein grafts has not been examined. This study assesses the in vitro isometric tension responses to specific adenosine receptor agonists (A1: R-phenyl-isopropyl-adenosine and A2: CSG-21680; 10–9 to 10–4 M) of common carotid external jugular vein bypass grafts (VG) in New Zealand White rabbits. These responses were compared to those obtained in the extemal jugular vein (JV) and in the common carotid artery (CA). All vessels were precontracted with prostaglandin F2a (10–5 M). Endothelialized and de-endothelialized vessels were examined. The A1 mediated relaxation in JV was endothelium-independent, whereas A2 mediated relaxation was endothelium-dependent. In CA, A1 mediated relaxation was partially endothelium-dependent, while A2 mediated relaxation was endotheliumindependent. In VG, Al activation induced endothelium-dependent vasoconstriction. Endothelial denudation restored Al mediated relaxation, but reduced compared to that of JV (max. 19 ± 9%). A2 relaxation in VG was endothelium-independent (max. 39 ± 4%). Primary cultures of arterial and vein graft SMC expressed both A1 and A2 receptors on northern blot analysis. There was, however, a marked reduction in the A1 affinity of the SMC from the VG compared to the arterial SMC (40.9 ± 1.3% arterial binding). This study, therefore, demonstrates that there is a substantial change in the adenosine mediated vasoreactivity in VG compared to JV, due to a change in endothelial A1 receptor mediated response from relaxation to constriction, coupled with a decreased affinity of the A1 receptors on the VG SMC. The adenosine responses of the VG are also different from CA. Therefore, the endothelial celis of VG appear to be unique, in that functionally they neither maintain a venous phenotype nor acquire an arterial phenotype in response to adenosine.