Immunocytochemical determination of cell type and proliferation rate in human vein graft stenoses

PURPOSE: Vascular reconstructions are prone to fail as a result of the development of stenotic lesions, which have historically been attributed to myointimal hyperplasia. In animal models, these lesions are associated with marked proliferative smooth muscle cell (SMC) response to vascular injury. However, recent studies using sensitive immunocytochemical techniques in human lesions have generally failed to detect significant cellular proliferation. To clarify the role of cellular proliferation in humans, we characterized the cellular composition and proliferative index of 14 early infrainguinal vein graft stenoses. METHODS: All infrainguinal vein grafts at our institution are prospectively enrolled in a duplex surveillance protocol, the details of which have been previously reported. Among 98 grafts placed within the last year, 11 patients were identified with 14 progressive, focal, high-grade lesions that met previously established threshold criteria for prophylactic revision to prevent graft thrombosis. Lesions were first detected from 1 week to 7 months after surgery and were removed and replaced with segmental interposition grafts (1.5 to 10 months). Freshly excised lesions were placed in Methyl Carnoy's fixative, paraffin embedded, and serially sectioned. The cellular composition of each lesion was determined with cell-specific immunochemical reagents: alpha SMC actin, von Willebrand factor (endothelial cell), CD 68 (macrophage), and CD 45RB (monocyte). Actively proliferating cells were identified using antibody to proliferating cell nuclear antigen (PCNA). The identity of PCNA-positive cells was determined by double-label immunocytochemical staining, and the proliferative index (PCNA-positive cells/total cells x 100) was calculated by computer-assisted counts of representative gridded cross-sections of each lesion. RESULTS: All excised lesions demonstrated marked thickening with severe luminal encroachment and were highly cellular, with a predominance of alpha SMC actin+. Endothelial cells on the blood flow surface were present to a variable degree, and seven lesions exhibited striking numbers of macrophages and monocytes. The latter cell types were most abundant near microvessels in the deep neointima and adventitia. Active cellular proliferation was identified primarily in SMCs, with a mean PCNA index of 1.34%. However, significant PCNA reactivity was not limited to SMCs, but was also identified in macrophages and monocytes, particularly in lesions greater than 3 months old. CONCLUSIONS: Previous immunocytochemical studies of human coronary restenosis atherectomy specimens have generally detected low rates of cellular proliferation (0.5%), but these lesions may not truly represent myointimal hyperplasia, rather a mixture of atherosclerosis, thrombosis, and "restenosis." In contrast, the present study of early human vein graft lesions detected by duplex surveillance indicates that significant cellular proliferation occurs, although rates are lower than those obtained in animals such as the rat carotid injury model. In addition, although SMCs are the predominant proliferating cell type in human vein grafts, our identification of proliferating monocytes and macrophages raises the question of the contribution of an inflammatory component to the development of human lesions. The present study represents the first report of PCNA determination in a series of human infrainguinal vein grafting procedures.     

Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein

Neointimal formation involving smooth muscle cell (SMC) migration and proliferation is a common feature of atherosclerosis, restenosis after angioplasty, and vein graft intimal thickening. Extracellular matrix remodeling by metalloproteinase (MMP) enzymes is an essential component of neointimal formation and therefore MMPs are a potential target for localized gene therapy. To evaluate this concept using human tissue, we used the highly reproducible organ culture model of neointimal formation in human saphenous vein to investigate the effect of adenovirus-mediated gene transfer of tissue inhibitor of metalloproteinase 1 (TIMP-1) and the bacterial LacZ gene (RAd35) as a control. Incubating veins with 100 microl of RAd35 (1.2 x 10(10) pfu/ml) led to expression of LacZ in 39 +/- 7% of surface cells but had no effect on SMC proliferation, migration, or neointimal formation. Similar infection with RAdTIMP-1 increased explanation of TIMP-1 in surface cells and significantly inhibited neointimal formation and SMC migration after 14 days by 54% and 78%, respectively (n = 6, p < 0.05 Student's paired t test). No effect on SMC proliferation or deleterious effect on cell viability was observed. A specific MMP inhibitory effect was detected using in situ zymography. These data confirm the importance of MMPs in neointimal formation and highlight the potential for application of TIMP gene therapy.     

Locally applied antisense oligonucleotide to proliferating cell nuclear antigen inhibits intimal thickening in experimental vein grafts

This study examines the effect of antisense oligonucleotide to proliferating cell nuclear antigen (PCNA) on the formation of vein graft intimal hyperplasia in vivo, using localized administration. Twenty-four New Zealand white rabbits had a right carotid interposition bypass graft using the external jugular vein and were sacrificed on the 28th postoperative day. To determine the effect of PCNA on the development of intimal hyperplasia, 6 animals had their grafts coated with a pluronic gel containing 18 base antisense oligonucleotide to PCNA (1 mg/ml), 6 received a pluronic gel containing an 18 base nonsense oligonucleotide (1 mg/ml), and 12 animals were controls (6 with and 6 without pluronic gel). These grafts were harvested for morphology and videomorphometry. There was no change in the intimal thickness between the control and gel-treated groups. (70 +/- 4 microm versus 72 +/- 4 microm; mean +/- s.e.m.; p = ns). The presence of nonsense oligonucleotide had no further effect. Antisense PCNA produced a 26% decrease in intimal thickness to 50 +/- 4 microm in the treated vein grafts (p < 0.03) without a change in medial thickness. This study shows that a local single application of antisense oligonucleotide to PCNA will reduce the intimal hyperplasia in experimental vein grafts over 28 days.     

Proliferative activity of gastric cancer assessed by immunostaining for proliferating cell nuclear antigen

The growth activity of 107 gastric carcinomas was assessed by immunohistochemical staining for formalin-fixed, paraffin-embedded tissue with a monoclonal antibody against proliferating cell nuclear antigen (PCNA). When the tumor doubling times (Tds) of 10 patients were estimated from the serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9, there was an inverse correlation between the Tds and PCNA labeling index (LI) at P = 0.055. Flow-cytometric analysis was carried out by double staining for PCNA and DNA using fresh materials from 14 patients. The PCNA-positive cell fraction revealed by flow cytometry showed a good linear correlation with PCNA LI in routinely stained tissue. The LI of well-differentiated adenocarcinoma was significantly higher than that of the poorly differentiated type. When the LI was analyzed in well- or poorly differentiated adenocarcinoma, the value was significantly higher in the well-differentiated type with hepatic metastasis and in the poorly differentiated type with lymph node metastasis.     

Expression of proliferating cell nuclear antigen in corneas kept in long term culture

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.     

Calcification at the posterior pole in scleritis. A case report

To explore the expression of proliferating cellnuclear antigen (PCNA) and tumor suppressor gene P53 product in mucoepidermoid carcinoma of the salivary glands, we studied 41 cases by immunohistochemical method. All of the cases were PCNA positive. The positive index, distribution pattern and cellular staining intensity for PCNA expression were correlated with carcinoma grade. P53 protein expression was found in 17 (41.5%) of the 41 cases; among them the expressions were weak in 9 cases. These results indicate that positive index, distribution pattern and cellular staining intensity of PCNA expression may be served as an indicator for evaluating the differentiating degree of the tumor.     

Marimastat inhibits neointimal thickening in a model of human vein graft stenosis

BACKGROUND: There is now accumulating evidence that matrix metalloproteinases (MMPs), the physiological mediators of matrix deposition and degradation, play an important role in the development of intimal hyperplasia following arterial bypass. This study investigated the effect of marimastat, an orally active specific MMP inhibitor, on neointima formation in cultured human saphenous vein. METHODS: Segments of human saphenous vein obtained from ten patients undergoing arterial bypass surgery were cultured for 14 days in serum-supplemented RPMI medium (controls) or in control medium supplemented with marimastat at three different concentrations (treatment groups). Following culture, half of each segment was prepared for histological examination and MMPs were extracted from the other half for gelatin zymography. RESULTS: Marimastat inhibited neointimal thickening in a concentration-dependent manner; inhibition was significant at 10(-5) and 10(-6) mol/l (P=0.006). This observation was paralleled by a significant reduction in the levels of MMP-2 and MMP-9 in the tissues. CONCLUSION: Marimastat significantly reduced neointimal thickening in this laboratory model. MMP inhibitors may offer a potential therapeutic strategy in the prevention of intimal hyperplasia.     

New insights for dinucleotide backbone binding in conserved C5''-H . . . O hydrogen bonds

OBJECTIVE: Although a number of pharmacologic agents have been shown to reduce intimal hyperplasia in animal models of restenosis, to date no systemic agent has conclusively been shown to be effective in humans. Recently, considerable attention has been directed towards endothelin (ET), a potent vasoconstrictor and a powerful mitogen for vascular smooth muscle cells, as a mediator of intimal hyperplasia. Endothelin-1 has been shown to be mitogenic for human saphenous vein smooth muscle cells, and expression also is elevated in human vein graft stenosis. The aim of this study was the investigation of whether ET receptor antagonists can attenuate neointima formation in a laboratory model of vein graft intimal hyperplasia and the determination of whether the effects are mediated by a specific ET receptor subtype. METHODS: We used an organ culture of human saphenous vein, a well-validated model of vein graft intimal hyperplasia. Paired segments of human long saphenous vein were cultured with and without the following antagonists: bosentan, a nonselective ET receptor antagonist; BQ 123, a specific endothelin-A antagonist; or BQ 788, a specific endothelin-B (ETB) antagonist. After 14 days in the culture, the segments were fixed and processed and the sections were immunostained to facilitate the measurements of neointimal thickness with a computerized image analysis system. RESULTS: The nonselective antagonist bosentan and the ETB selective antagonist BQ 788 significantly reduced neointima formation by 70% (P = .001) and 50% (P = .03), respectively, but the ETA antagonist BQ 123 had no significant effect on the reduction of neointima formation (P = 1.0). CONCLUSION: The results of this study imply an important role for ET as a mediator of human vein graft intimal hyperplasia and imply further that a specific ETB antagonist may have a therapeutic potential for the prevention of vein graft stenosis.     

Effort and achievement in national family planning programmes

Cell proliferation of 174 specimens obtained from the primary gastric cancers using endoscopic biopsy was investigated by immunohistochemical analysis with the monoclonal antibody PC10, which recognizes a proliferating cell nuclear antigen (PCNA) in formalin-fixed and paraffin-embedded material. All the examined samples showed nuclear staining for PCNA in cancer cells. The investigation was to test the correlation between PCNA labeling and lymph node metastasis. DNA aneuploidy was often encountered in tumors with nodal involvement and lymphatic invasion. The logistic regression analysis identified PCNA labeling rates (LRs), tumor size, and macroscopic type as independent significant factors for lymph node metastasis. When the PCNA LRs and clinicopathologic parameters were entered into the Cox regression analysis, PCNA LRs and DNA ploidy emerged as independent significant prognostic factors. In addition, combination assay of PCNA LRs and DNA ploidy yielded a powerful prognostic indication for patients with gastric cancer.     

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

PURPOSE: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS: Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION: When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.     

Expression of proliferating cell nuclear antigen (PCNA) in premalignant lesions of laryngeal epithelium

It is important to study the proliferative activity of cells in the premalignant lesions of laryngeal epithelium. Using PC-10, an antibody to PCNA and a standard immunohistochemical staining, we examined 11 cases of simple hyperplasia of epithelium (SHE), 32 cases of atypical hyperplasia of epithelium (AHE) and 42 cases of laryngeal squamous cell carcinoma (LSCC) for expression of PCNA, a protein associated with DNA polymerase delta and DNA replication. The results revealed that the PCNA indices in SHE, AHE and LSCC were 9.57%, 27.33% and 68.05%, respectively. The PCNA indices were 13.79%, 30.84% and 39.94%, in the mild, moderate and severe AHE, respectively. There were significant differences among the SHE, AHE and LSCC. A good correlation was found among different degrees of AHE. The pattern and location of PCNA positive cells in the intraepithelium had diagnostic importance. PCNA might provide a useful tool for studying cell proliferation in situ under normal and pathological conditions.     

Haemodynamic effects of eating: the role of meal composition

I have investigated 84 endometrial specimens (from 15 cases of normal endometrium, 20 cass of hyperplasia and 49 cases of endometrial carcinoma) to determine the relationship between three proteins (proliferating cell nuclear antigen (PCNA), p53 gene product and c-erB-2 gene product) and endometrial carcinoma by immuno-histochemical staining. In 49 cases of endometrial carcinoma, the positive rates for PCNA, p53 protein (mutant type) and c-erbB-2 protein were 65.3%, 59.2% and 22.4%. I could not find the expression of p53 protein besides endometrial carcinoma. And I could find the expression of c-erbB-2 protein in 11 cases of endometrial carcinoma and 1 case of atypical hyperplasia, but not in normal endometrium. p53 protein was more common in such a case, as with lymphnode metastasis, deep myometral invasion and undifferentiated adenocarcinoma. c-erbB-2 was also more common in a case with deep myometrial invasion. In conclusion, PCNA, p53 protein and c-erbB-2 protein are related to the proliferation of endometrial carcinoma. So they can be useful factors in making the prognosis.     

New histopathologic data of prognostic value in extra-adrenal paragangliomas. Study of 9 cases

BACKGROUND: The biological behavior of paragangliomas is difficult to evaluate by classic histological criteria thus justifying the use of immunohistochemical markers as prognostic factors. METHODS: Nine extra-adrenal paragangliomas (three jugulo-tympanic, four carotid-body tumors, and two retroperitoneal) were studied by conventional histological criteria, and also by chromogranin A and neuron-specific enolase (NSE) immunohistochemical staining for the study of chief cells, and S-100 as a marker of sustentacular cells. The rate of cell proliferation was studied by the proliferating cell nuclear antigen (PCNA). The correlation between these parameters and the clinical evolution of the neoplasms, which were classified as benign, locally aggressive, and malignant (with metastasis), were also analyzed. RESULTS: The atypia and the mitotic rate did not correlate with the behavior of the tumor. Less immunostaining with the anti-S-100 and anti-chromogranin A antibodies was observed in the malignant paragangliomas and in those which were locally aggressive. In the benign tumors the proliferative rate (PCNA) oscillated between 0.7% and 3.7%, and 40 or less PCNA positive cells were counted in 10 high-power field (HPF) (40x). In malignant and locally aggressive tumors the proliferative rate was 5% or more, with 60 or more cells that were positive for PCNA being found in 10 HPF. CONCLUSIONS: The histopathologic signs implying worse prognosis in extra-adrenal paragangliomas are a decrease in chromogranin A and S-100 immunoreactivity and a rate of cell proliferation of 5% or greater, or a number of cells stained for proliferating cell nuclear antigen greater than 50 in 10 high-power field.     

A randomized double-blind trial of ondansetron alone versus in combination with dexamethasone versus in combination with dexamethasone and lorazepam in the prevention of emesis due to cisplatin-based chemotherapy

Sixty-five cases of invasive breast cancer < or = 1 cm. in largest diameter (pT1a-b) were studied retrospectively using immunohistochemical staining with PC10, a monoclonal antibody to proliferating cell nuclear antigen (PCNA). The percentage of PC10 positive tumor cells was closely related to histological grading. No association was found between PC10 score and nodal status. ER-ICA was performed on 42 cases and showed no correlation with PC10 staining. The clinical behaviour of these tumors was excellent, with 5-year survival rates overall of 96% (90% disease free survival), and apparently unrelated to histological type and grade, nodal involvement and hormonal receptor status. The prognostic value of PCNA labeling rates remains nuclear in breast cancer of minimal size as well as in larger ones.     

Endothelin-1 and cell proliferation in lung organ cultures. Implications for lung allografts

Endothelin-1 (ET-1) is found in bronchoalveolar lavage fluid in patients following lung transplantation. ET-1 causes contraction of isolated pulmonary vessels and bronchi and stimulates proliferation of smooth muscle cells in culture. Therefore, ET-1 could contribute to the smooth muscle hyperplasia and stromal proliferation seen in chronic rejection of lung allografts. Experiments were designed to determine whether (1) ET-1 stimulates proliferation of pulmonary tissue, (2) proliferation is increased in rejecting allotransplanted lungs, (3) endothelin-A receptors mediate the proliferative response, and (4) ET-1 is produced by activated infiltrating immunocompetent cells. Lung organ cultures were prepared from unoperated dogs and dogs with rejecting single lung allografts. Incubation of organ cultures from unoperated dogs with ET-1 (10(-9) to 10(-7) M)) increased positive staining for proliferation cell nuclear antigen (PCNA) in lung parenchyma. PCNA staining was not decreased by the endothelin-A antagonist BQ123 (10(-6) M). In addition, immunostaining for endothelin-B receptors was present in sections of unoperated but not rejecting lungs. PCNA staining in lung cultures from rejecting allotransplanted dogs was significantly greater than that from unoperated dogs. Positive immunohistochemical staining for ET-1 was found in mononuclear cells infiltrating rejecting transplanted lungs. In conclusion, exogenous ET-1 is mitogenic in lung organ cultures through receptors other than endothelin-A. Proliferation in rejecting transplanted lungs is increased compared with unoperated lungs. Mononuclear cells may be a source of endothelin-1 in the rejecting lung. ET-1, therefore could, in synergism with other cytokines, contribute to acute and chronic pathological changes seen in pulmonary rejection.     

Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumour cell proliferation in human epidermoid lung carcinoma

Vascular endothelial growth factor (VEGF) expression, vascularisation and tumour cell proliferation were analysed in 91 human epidermoid lung carcinomas using immunohistochemistry. A polyclonal anti-VEGF antibody was used for VEGF expression, a polyclonal antibody directed against human von Willebrand factor (factor VIII) to identify blood vessels and the proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells. Positive staining for VEGF was obtained in 54 out of 91 cases (59%), the number of blood vessels varied from zero to 64 counts (mean 9.4) and the proportion of PCNA-positive cells varied from 1.3% to 72.1% (mean 25.2%). The mean PCNA labelling index and mean microvessel count in VEGF-positive tumours were significantly higher than those in VEGF-negative tumours (Wilcoxon rank sum test, P<0.0001; p<0.05). In addition, PCNA labelling index significantly increased with increasing VEGF expression (Jonckheere test, P<0.0001). In contrast, no association was found between PCNA labelling index and tumour vascularity (r=0.07, P=0.48). The close correlation of VEGF expression with tumour cell proliferation and microvessel density suggests that VEGF acts both as an autocrine growth factor and as stimulator for angiogenesis. However, tumour cell proliferation and microvessel growth and/or density may be regulated by separate mechanisms.     

Temporal expression of c-fos mRNA following balloon injury in the rat common carotid artery

OBJECTIVE: Restenosis is a common problem which limits the effectiveness of percutaneous transluminal coronary angioplasty (PTCA). The cellular mechanisms of restenosis appear to involve smooth muscle cell (SMC) migration to the neointima in response to mitogens and growth factors, resulting in proliferation and deposition of cells in the lumen of the vessel. An antibody directed against PDGF attenuates this response in the rat. Thus, signaling cascades induced by growth factors including PDGF may be important targets for therapeutic intervention. METHODS: Since a number of growth factors activate c-fos via the p21-ras signaling pathway, we examined c-fos expression in a time course experiment involving restenotic lesions in rat carotid arteries. Sections of arteries collected at 1, 3, 7, 14 and 28 days following balloon injury were hybridized using a fluorescein-labeled RNA probe to c-fos. Immunohistochemistry was performed with antibodies to proliferating cell nuclear antigen (PCNA) and alpha-smc actin to characterize cellular constituents of the neointima, and detect any correlation between fos expression and PCNA localization. RESULTS: Expression of c-fos was low at day 1. By day 3, the media and adventitia were positively stained. At days 7 and 14, most cells in the neointima were labeled. By day 28, c-fos was expressed mainly in scattered cells along the luminal surface. Control sections revealed little labeling and confirmed specific staining by the antisense strand, PCNA localization and c-fos expression were similar at days 1, 3, 7 and 28, but at day 14 c-fos was expressed throughout the lesion, with PCNA localized mainly along the luminal edge. The majority of the cells making up the neointima stained rather intensely for alpha-smc actin, identifying them as SMCs. CONCLUSIONS: Results of these experiments indicate that, while c-fos expression correlates with lesion formation, it may be associated with a cellular process distinct from proliferation in this model.     

Fibrin glue containing fibroblast growth factor type 1 and heparin with autologous endothelial cells reduces intimal hyperplasia in a canine carotid artery balloon injury model

PURPOSE: Intimal hyperplasia plagues all types of vascular intervention. Early confluent re-endothelialization may attenuate the smooth muscle cell (SMC) proliferative response. We previously reported that fibroblast growth factor type 1 (FGF-1) and heparin at relative concentrations of 10 ng/ml:250 U/ml delivered in a fibrin glue (FG) suspension can selectively stimulate endothelial cells (EC) and inhibit SMC proliferation in cell culture. This current study evaluates this surface treatment with and without seeded autologous ECs on intimal hyperplasia in a canine carotid artery balloon injury model. METHODS: Twenty-nine adult dogs underwent bilateral balloon injury to a 6 cm segment of their carotid arteries. The injury resulted in a reproducible removal of the intima and 4 to 6 medial lamellae. Nine dogs were used in part I to determine the percent retention of FGF-1 and EC when applied in a FG suspension to the balloon-injured carotid arteries. Part 2 used the remaining 20 dogs to determine the effect of this surface treatment on intimal hyperplasia. In 10 group I dogs, FG (fibrinogen 32.1 mg/ml and thrombin 0.32 U/ml) containing FGF-1 (11 ng/ml) and heparin (250 U/ml) was applied to the luminal surface of one carotid artery, whereas the contralateral carotid artery underwent balloon injury alone. In 10 group II dogs, an identical FG preparation with FGF-1 and heparin was applied to the surface of one carotid artery, whereas the contralateral carotid artery received FG/FGF-1/heparin that also contained autologous ECs (P3; 5 x 10(4) to 10 x 10(4) cells/cm2). Five dogs from both group I and group II were killed at 10 days and the remaining 10 dogs at 30 days. Histologic analysis and computerized morphometric analysis were used to determine intimal and medial thickness and area, percent endothelialization, and medial SMC proliferative rate. RESULTS: There was no measurable neointima in any 10-day dog. There was no difference in neointimal area between the treatments in group I 30-day dogs. There was a significant decrease in maximal neointimal area, intima/media thickness ratio, and intima/media area ratio in group II 30-day dogs that were treated with FG/FGF-1/heparin plus EC. There was an insignificant increase in percent EC coverage and an insignificant decrease in medial SMC proliferative rate in group II 10-day dogs treated with FG/FGF-1/heparin plus EC. CONCLUSIONS: In this canine carotid model, FG with FGF-1 and heparin did not induce significant intimal or medial thickening after 10 or 30 days when compared with vessels that were only balloon-injured. The seeding of autologous ECs within the FG/FGF-1/heparin suspension caused a reduction in neointima formation with no concomitant medial thickening 30 days after injury. The use of FG to locally deliver FGF-1 and ECs may have clinical relevance in the inhibition of intimal hyperplasia.     

Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins

Metalloproteinases (MMPs) are implicated in neointima formation and hence vein graft failure. Gene transfer to elevate local levels of tissue inhibitor of metalloproteinases (TIMPs) is therefore a potential treatment. In this study, we have used lumenal application of a replication-defective recombinant adenovirus to overexpress TIMP-2 and observe the effects on neointimal thickening in a well characterised human saphenous vein organ culture model. Increased TIMP-2 expression was localised to lumenal surface cells but nevertheless increased total functional TIMP-2 secretion after 14 days culture from 4.0 +/- 2.0 to 21.8 +/- 2.9 ng/mg wet weight/day (P < 0.05, n = 3). In situ zymography revealed a marked inhibition of gelatinolytic activity by TIMP-2 gene transfer throughout the vein segments. Neointima formation and neointimal cell numbers were reduced 79% and 71%, respectively (P < 0.05; n = 8). TIMP-2 overexpression had no effect on smooth muscle cell proliferation, secretion of pro-MMP-2 or -9 and did not inhibit the processing of pro-MMP-2 to its active form. Our data indicate that TIMP-2 overexpression reduces neointimal thickening, primarily by inhibiting MMP activity and hence smooth muscle cell migration.