A pilot study of pi-class glutathione S-transferase expression in breast cancer: correlation with estrogen receptor expression and prognosis in node-negative breast cancer.

PURPOSE AND METHODS: Previous studies have indicated that RNA levels for pi-class glutathione S-transferase (GST pi), a phase II, drug-metabolizing enzyme, were inversely related to estrogen receptor (ER) and progesterone receptor (PR) levels in human breast tumors. Because GST pi also is expressed in normal breast epithelium, an immunohistochemical assay that uses affinity-purified polyclonal antibodies to GST pi was developed to examine the possible relationship between GST pi expression in breast cancer cells and hormone receptor expression, as well as prognosis, in patients with primary breast cancer. RESULTS: A strong inverse correlation between GST pi expression and ER (two-sided P [P2] = .002) and PR status (P2 = .023) was found in our study of 189 patients with primary breast cancer. GST pi expression was not related to tumor size, nodal metastasis, nuclear grade, histology, or age of the patient. In node-negative breast cancer (n = 72), increased GST pi expression was associated with decreased disease-free survival (DFS) and overall survival (OS). When GST pi expression was divided into categories of negative (no GST pi-positive tumor cells), intermediate (1% to 70% GST pi-positive tumor cells), and high (> 70% GST pi-positive tumor cells), the relative risk of tumor recurrence in patients with node-negative breast cancer was increased 3.39-fold for each successive category of expression (P2 = .0045; 95% confidence interval, 1.46 to 7.87) and the relative risk of death was increased 4.49-fold for each successive category (P2 = .0003; 95% confidence interval, 2.02 to 10.42). The actuarial 5-year OS was 100%, 79%, and 51%, and the DFS was 94%, 77%, and 44%, for the negative, intermediate, and high tumor groups, respectively. Among the factors studied in multivariate analysis (ER status, PR status, nuclear grade, and tumor size), GST pi expression was the factor that most accurately predicted shorter DFS and OS in node-negative patients. CONCLUSION: GST pi expression is inversely related to hormone receptor status in breast cancer. This pilot study also suggests that increased GST pi expression may be an important predictor of early recurrence and death in node-negative breast cancer patients that merits additional investigation.

Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs.

OBJECTIVE: In preparation for clinical trials of basic fibroblast growth factor (bFGF) to treat ischemic heart disease, we sought to identify a clinically feasible method of bFGF administration. BACKGROUND: Basic FGF has been shown to promote collateral development after experimentally induced coronary occlusion; however, methods of bFGF delivery that have been shown to be effective in previous investigations would not be practical for clinical use. METHODS: Four randomized, blinded, controlled investigations were conducted independently and sequentially in an established canine model. For all studies, dogs underwent operative placement of proximal left circumflex coronary artery ameroid constrictors. The four investigational regimens included: 1) bFGF by central venous bolus injection, 1,740 microg/day for one, two or seven days; 2) bFGF by intravenous infusion, 100 microg/kg body weight per day for seven days; 3) bFGF by pericardial instillation, 2,000 microg/day for 7 days; and 4) bFGF by intracoronary injection (Judkin's technique), 100 microg/kg per day for one or two days. Each substudy included a contemporaneous vehicle control group. Collateral perfusion (microspheres) was assessed during maximal coronary vasodilation during the first month after ameroid placement. RESULTS: Maximal collateral perfusion in dogs that received intracoronary bFGF for two days exceeded that of concurrent control dogs by 31% (p < 0.01). Perfusion was not increased in dogs that received single-dose intracoronary bFGF. Basic FGF administration by central venous bolus injection, intravenous infusion and pericardial injection failed to enhance collateral perfusion. CONCLUSIONS: Administration of bFGF by the intracoronary route, an intervention that is feasible in patients, augments collateral development in dogs. These data provide a rationale for clinical testing of intracoronary bFGF in ischemic heart disease.

Basic fibroblast growth factor in patients with intermittent claudication: results of a phase I trial.

OBJECTIVES: This phase I study was designed to evaluate the safety, tolerability and pharmacokinetics of intra-arterial basic fibroblast growth factor (bFGF) in patients with atherosclerotic peripheral arterial disease (PVD) and intermittent claudication. We also assessed the effects of basic fibroblast growth factor (bFGF) on calf blood flow as a measure of biologic activity. BACKGROUND: Preclinical studies have shown that bFGF, an angiogenic peptide, promotes collateral development in animal models of myocardial and hind limb ischemia. The safety and efficacy of bFGF in patients is unknown, and early clinical trials are underway in coronary and peripheral arterial disease. METHODS: A double-blind, placebo-controlled, dose-escalation trial was conducted in patients with claudication demonstrating ankle/brachial index <0.8. Patients were randomly assigned to placebo (n = 6), 10 microg/kg of bFGF (n = 4), 30 microg/kg of bFGF once (n = 5) and 30 microg/kg of bFGF on two consecutive days (n = 4). Study drug was infused into the femoral artery of the ischemic leg. Detailed safety information including retinal photography for neovascularization were obtained through one year. Calf blood flow was measured with strain gauge plethysmography in the two higher dose treatment groups and in four placebo patients at baseline, one month and three to seven months after treatment. RESULTS: Intra-arterial bFGF was safe and well-tolerated. The half-life was 46 +/- 21 min. Calf blood flow increased at one month by 66 +/- 26% (mean +/- SEM) and at six months by 153 +/- 51% in bFGF-treated patients (n = 9, p = 0.002). Flow did not change significantly in the placebo group. CONCLUSIONS: In this initial randomized, double-blind, placebo-controlled trial in patients with atherosclerotic PVD and claudication, bFGF was well-tolerated. The data suggest a salutary biologic effect, and initiation of phase 2 trials is warranted.

Effect of basic fibroblast growth factor on myocardial angiogenesis in dogs with mature collateral vessels.

OBJECTIVES: We sought to evaluate the potential of basic fibroblast growth factor (bFGF) to enhance coronary collateral perfusion in dogs with chronic single-vessel coronary occlusion. A secondary goal was to examine whether the salutary effects of bFGF treatment, previously proved effective in the short term, would be maintained in the long term (6 months). BACKGROUND: bFGF, an angiogenic growth factor, is currently the subject of a Phase I trial in patients with ischemic heart disease. It has been shown to promote collateral development in dogs with progressive coronary occlusion when given during the period of natural collateralization. The effect of bFGF on quiescent collateral vessels, a subject of significant clinical importance, is uncertain. METHODS: Dogs were subjected to ameroid-induced occlusion of the left circumflex coronary artery and randomized to bFGF (1.74 mg/day for 7 days), a regimen previously proved effective, or to saline solution. Maximal collateral perfusion was assessed 6 months later, and the dogs were reassigned to a course of bFGF or saline solution. Collateral perfusion was reevaluated after the second treatment course. RESULTS: At 6 months, collateral function was identical in the groups treated initially with bFGF and saline solution. The subsequent course of bFGF did not induce further collateralization. CONCLUSIONS: Although we previously demonstrated the salutary effects of this bFGF regimen in the short term (5 weeks), collateral flow in control dogs reached parity with that of bFGF-treated dogs after 6 months. bFGF did not induce further collateralization in dogs with mature collateral vessels, underscoring the priming role of ischemia for bFGF-induced collateral development.

Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis.

OBJECTIVE: Angiogenic peptides like VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) have entered clinical trials for coronary artery disease. Attempts are being made to devise clinically relevant means of delivery and to effect site-specific delivery of these peptides to the cardiac tissue, in order to limit systemic side-effects. We characterized the response of the pericardium to delivery of a replication-deficient adenovirus carrying the cDNA for AdCMV.VEGF165, and assessed the effect of pericardial VEGF165 on myocardial collateral development in a canine model of progressive coronary occlusion. METHODS: Ameroid constrictors were placed on the proximal left circumflex coronary artery of mongrel dogs. Ten days later, 6 x 10(9) pfu AdCMV.VEGF165 (n = 9). AdRSV.beta-gal (n = 9), or saline (n = 7) were injected through an indwelling pericardial catheter. Transfection efficiency was assessed by X-gal staining. Pericardial and serum VEGF levels were measured serially by ELISA. Maximal myocardial collateral perfusion was quantified with radiolabeled or fluorescent microspheres 28 days after treatment. RESULTS: In AdRSV.beta-gal-treated dogs, there was extensive beta-gal staining in the pericardium and epicardium, with minimal beta-gal staining in the mid-myocardium and endocardium. Pericardial delivery of AdCMV.VEGF165 resulted in sustained (8-14 day) pericardial transgene expression, with VEGF levels peaking 3 days after infection (> 200 ng/ml) and decreasing thereafter. There was no detectable increase in serum VEGF levels. Maximal collateral perfusion, a principal correlate of collateral development and angiogenesis, was equivalent in all groups. CONCLUSION: Adenoviral-mediated gene transfer is capable of inducing sustained VEGF165 expression in the pericardium; however, locally targeted pericardial VEGF delivery failed to improve myocardial collateral perfusion in this model.

Pharmacodynamics of basic fibroblast growth factor: route of administration determines myocardial and systemic distribution.

OBJECTIVE: We have shown that basic fibroblast growth factor (bFGF/FGF-2) enhances myocardial collateral development in a canine model of progressive coronary occlusion when delivered via the left atrial or intracoronary routes; however, we have found intravenous bFGF ineffective in the same model. Data on the fate and efficacy of intravenous bFGF are limited. We hypothesized that first pass lung uptake might limit myocardial bFGF availability after intravenous injection. We postulated that delivery of bFGF through the distal port of a wedged Swan Ganz catheter might circumvent this problem by restricting exposure of bFGF to a limited number of pulmonary binding sites. This study evaluated differential regional uptake of 125I labeled bFGF following bolus intravenous, Swan Ganz, left atrial, intracoronary, and pericardial delivery. METHODS: Mongrel dogs were used. Human recombinant bFGF, monoiodinated with 125I, was mixed with cold bFGF to a specific activity of 0.03 microCi/microgram. Approximately 100 micrograms/kg was injected per animal by the intravenous, left atrial, Swan Ganz, intracoronary, or pericardial route. Dogs were killed 15 min or 150 min later. The heart, lungs, liver, spleen, and kidneys were harvested and 125I activity was assessed. Immunohistochemical and pharmacokinetic studies were also performed. RESULTS: Serum half life of bFGF was comparable after intracoronary, intravenous and left atrial delivery (50 min); however, there were significant differences with regard to pharmacodynamics. After intracoronary administration, 3-5% of the total bFGF dose was recovered from the heart, with the peptide immunolocalized to the extracellular matrix and vascular endothelium. In contrast, only 1.3% of the injected bFGF was localized to the heart after left atrial administration and 0.5% was recovered after intravenous or Swan Ganz delivery. Pericardial administration resulted in substantial cardiac bFGF delivery; 19% was present at 150 min. Myocardial uptake was similar with Swan Ganz and intravenous delivery, suggesting that the administered dose did not saturate available pulmonary binding sites. CONCLUSIONS: These data predict efficacy of intracoronary, left atrial, and pericardial bFGF for myocardial angiogenesis, and a lack of efficacy after bolus intravenous and Swan Ganz administration.

Combined bromodeoxyuridine immunohistochemistry and Masson trichrome staining: facilitated detection of cell proliferation in viable vs. infarcted myocardium.

Cells in the S-phase of the cell cycle can be identified in tissue sections by immunohistochemical localization of the thymidine analogue bromodeoxyuridine (BrdU). Generally, a single counterstain is used to visualize the underlying tissue; however, interpretation of morphologic detail is often difficult. We have utilized BrdU to localize proliferating cells in myocardium exposed to angiogenic mitogens. To facilitate identification of labelled nuclei in the context of infarcted vs. viable myocardium, BrdU immunohistochemistry was followed by a modified Masson trichrome stain. The time of exposure to the counterstains and the wash protocol were re-revised, permitting clear identification of the labelled brown nuclei against a background of red viable myocardium vs. blue infarct. The combined technique also provides color contrast suitable for computer-based image analysis.

Neointimal proliferation in canine coronary arteries. A model of restenosis permitting local and continuous drug delivery.

BACKGROUND: A number of experimental preparations have been used to elucidate the pathophysiology of restenosis after percutaneous transluminal coronary angioplasty; however, few models have been advanced that address restenosis in coronary arteries, and none provides an effective means of continuous local drug delivery. In this report, we describe a model of restenosis in coronary arteries with the provision for local, continuous delivery of cytotoxic and/or anti-proliferative agents. EXPERIMENTAL DESIGN: An ameroid constrictor was placed on the left circumflex coronary artery of 17 normocholesterolemic dogs. One month later, after substantial collateral development had ensued, a segment of the left circumflex coronary artery distal to the ameroid was mechanically compressed using surgical forceps for 10 (N = 4), 15 (N = 4), 20 (N = 2), or 30 minutes (N = 5). In two dogs, an indwelling left circumflex catheter and implanted pump maintained a continuous infusion of saline at the injury site. In addition, the pump side port provided transcutaneous access for serial, selective coronary arteriography. The animals were maintained on a normal diet, without cholesterol or fat supplementation. RESULTS: Three weeks after vascular injury, significant neointimal proliferation was observed in all dogs that was morphologically similar to the proliferation seen after percutaneous transluminal coronary angioplasty in human coronary arteries. The extent of neointimal formation was linearly related to the duration of injury: neointimal/medial area ratios were 0.35 +/- 0.10, 0.46 +/- 0.10, 0.58 +/- 0.03, and 1.16 +/- 0.26 (mean +/- SE) after 10, 15, 20, and 30 minutes of mechanical compression injury, respectively. CONCLUSIONS: This model produces striking neointimal proliferation in the coronary arteries of normocholesterolemic dogs, morphologically similar to that seen in human coronary restenosis specimens. The model appears suitable to test the efficacy of agents with the potential to inhibit neointimal formation, providing continuous intracoronary drug delivery, as well as transcutaneous access for serial, selective arteriography.

Histological evaluation of the canine retinal vasculature following chronic systemic administration of basic fibroblast growth factor.

The purpose of the present study was to determine if prolonged systemic arterial administration of basic fibroblast growth factor (bFGF) at a dose sufficient to enhance collateral vessel formation in the ischaemic hearts of dogs would produce retinal neovascularisation in these same animals. Adult dogs (15-25 kg) were subjected to gradual occlusion of a coronary artery and randomised to receive 1 of 3 treatments via an indwelling left atrial catheter: (1) bFGF 1.74 mg/d, 5 d/wk for 63 d (n = 7); (2) bFGF 1.74 mg/d, 5 d/wk, for 35 d followed by physiological saline, 5 d/wk, for 28 d (n = 10); or (3) physiological saline, 5 d/wk, for 63 d (n = 10). After 63 d the retinal vasculatures from these dogs were isolated and examined for capillary varicosity, neovascularisation and other histopathological signs of angiopathy. All data were collected under masked conditions. The results suggest that chronic, systemic arterial administration of bFGF stimulates neovascularisation in the ischemic myocardium, but has no significant structural or vasoproliferative effect on the nonischaemic retina of the same animal.

Effects of chronic systemic administration of basic fibroblast growth factor on collateral development in the canine heart.

BACKGROUND: Recently we reported that intracoronary administration of basic fibroblast growth factor (bFGF), a potent angiogenic peptide, increases collateral blood flow in dogs subjected to progressive left circumflex coronary artery (LCx) occlusion. The aim of the present study was to examine the effect of systemically administered bFGF on collateral blood flow and to assess its pharmacokinetics and potential side effects. METHODS AND RESULTS: Forty-seven dogs were subjected to progressive ameroid-induced occlusion of the LCx, an intervention known to induce the development of collateral vessels. In phase I of the investigation, dogs were randomized to receive bFGF 1.74 mg/d (n = 10) or saline (n = 9) as a left atrial injection for 4 weeks. Relative collateral blood flow was assessed serially with radiolabeled microspheres in the conscious state during maximal coronary vasodilatation. Initiation of bFGF treatment was temporally associated with a marked acceleration of collateral development; however, collateral flow in control dogs improved toward the end of the study, approaching that of bFGF-treated dogs at the 38-day end point. Phase II of the investigation was a three-armed study of extended duration to determine whether bFGF caused a sustained increase in collateral function. Dogs were randomized to receive bFGF 1.74 mg/d for 9 weeks (n = 7), bFGF 1.74 mg/d for 5 weeks followed by placebo for 4 weeks (n = 11), or placebo for 9 weeks (n = 10). Relative and absolute collateral blood flow were assessed serially with microspheres during maximal coronary vasodilatation. Between the 10th and 17th days after ameroid placement, bFGF-treated dogs exhibited marked improvement in collateral flow such that maximal collateral conductance exceeded that of controls by 24% at the 5-week crossover point. Final collateral conductance was similar in dogs receiving bFGF for 5 and 9 weeks despite withdrawal of treatment in the former group. bFGF administration was associated with a 21% increase in final collateral conductance as well as a 49% increase in collateral zone vascular density. Prolonged bFGF administration was also associated with a decrease in arterial pressure, moderate thrombocytopenia, and moderate, reversible anemia. CONCLUSIONS: Systemic administration of bFGF enhanced collateral conductance in dogs with progressive single-vessel coronary occlusion. The beneficial effect of bFGF occurred primarily between the 7th and 14th days of therapy, and regression of collateral development was not noted after withdrawal of treatment. The present investigation provides impetus to the concept that collateral development can be enhanced pharmacologically-specifically by bFGF-raising the possibility that such an intervention might eventually be applied clinically.

Basic fibroblast growth factor enhances myocardial collateral flow in a canine model.

Basic fibroblast growth factor (FGF) is a multifunctional peptide that may play an integral role in angiogenesis associated with coronary collateral formation and myocardial infarct healing. We sought to determine the effects of exogenously administered basic FGF on collateral blood flow to ischemic myocardium. Ameroid constrictors were used to cause gradual occlusion of the left circumflex coronary artery in dogs. Basic FGF (110 micrograms, n = 9) or saline (n = 12) was given as a daily bolus injection directly into the collateral-dependent zone, beginning 10 days after placement of the Ameroid and continuing for 28 days. Collateral flow was assessed weekly as the ratio of collateral to normal zone (CZ/NZ) blood flow during maximal pharmacologically induced coronary vasodilation. The CZ/NZ increased in both treated and control dogs as a function of time; however, transmural collateral flow in basic FGF-treated dogs significantly exceeded that of control dogs by the second week of treatment. Final CZ/NZ blood flow ratios were 0.49 +/- 0.05 and 0.35 +/- 0.02 in the treated and control groups, respectively (means +/- SE, P = 0.0002). Treatment with basic FGF was also associated with significant increases in the numerical density of distribution vessels and endothelial cell DNA synthesis within the CZ. We also found that basic FGF had acute effects as a coronary vasodilator. Thus exogenous administration of basic FGF enhances maximal collateral blood flow in dogs with myocardial ischemia secondary to single-vessel coronary occlusion, an effect that is likely mediated through the direct angiogenic effects of the peptide, although its acute vasodilatory effects may also play a role.

Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is angiogenic in vitro and in vivo. It has been hypothesized that VEGF plays a role in myocardial collateral formation; however, the effects of VEGF on collateral flow to ischemic myocardium are unknown. METHODS AND RESULTS: We studied the effect of VEGF on collateral blood flow in dogs subjected to gradual occlusion of the left circumflex coronary artery (LCx). Beginning 10 days after placement of an LCx-constricting device, VEGF 45 micrograms (n = 9) or saline (n = 12) was administered daily via an indwelling catheter in the distal LCx, at a point just beyond the occlusion. Treatment was maintained for 28 days. Collateral blood flow was determined with microspheres 7 days before treatment, immediately before treatment (day 0), and 7, 14, 21, and 28 days into the treatment period. Collateral blood flow was quantified during chromonar-induced maximal vasodilation and expressed as a collateral zone/normal zone (CZ/NZ) ratio. Treatment with VEGF was associated with a 40% increase in collateral blood flow (final CZ/NZ blood flow ratios of 0.49 +/- 0.06 and 0.35 +/- 0.02 in the VEGF-treated and control groups, respectively, P = .0037) as well as an 89% increase in the numerical density of intramyocardial distribution vessels (> 20 microns diameter) in the CZ (6.6 +/- 1.4 versus 3.5 +/- 0.7 vessels/mm2 in VEGF-treated and control dogs, respectively, P < .05). CONCLUSIONS: We conclude that intracoronary VEGF enhances the development of small coronary arteries supplying ischemic myocardium, resulting in marked augmentation of maximal collateral blood flow delivery. These results demonstrate the feasibility of pharmacological enhancement of collateral growth and suggest a new therapeutic approach for the treatment of myocardial ischemia.

Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury.

BACKGROUND: We have shown that the angiogenic peptides basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) enhance canine coronary collateral development when administered for > or = 4 weeks. bFGF, a pluripotent mitogen of mesodermally derived cells, could theoretically exacerbate neointimal smooth muscle cell hyperplasia, a fundamental component of atherosclerosis. VEGF, an endothelial cell-specific mitogen and vascular permeability factor, could have deleterious effects related to vascular hyperpermeability. The present investigation had two aims: (1) to ascertain whether brief (7-day) systemic arterial treatment with bFGF or VEGF would improve myocardial collateral perfusion and (2) to determine whether these peptides induce neointimal accumulation in vivo. METHODS AND RESULTS: Dogs were subjected to ameroid-induced occlusion of the left circumflex coronary artery and randomized to bFGF 1.74 mg (n = 9), VEGF 0.72 mg (n = 9), or saline (n = 10) as a daily left atrial bolus (days 10 to 16). Additional dogs were randomized to VEGF 0.72 mg (n = 6) or saline (n = 5); however, treatment was delayed by 1 week. Coincident with the institution of treatment, all dogs underwent balloon denudation injury of the iliofemoral artery. bFGF markedly increased maximal collateral flow but did not exacerbate neointimal accumulation. VEGF had no discernible effect on maximal collateral flow, but it exacerbated neointimal thickening after vascular injury. CONCLUSIONS: Short-term treatment with bFGF enhanced collateral development without increasing neointimal accumulation at sites of vascular injury. Although VEGF did not increase collateral development as administered in this study, it significantly exacerbated neointimal accumulation. These data provide support for the clinical investigation of bFGF in selected patients with ischemic heart disease.