Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries.

Injury to atherosclerotic arteries induces the expression of growth regulatory genes that stimulate cellular proliferation and intimal formation. Intimal expansion has been reduced in vivo in nonatherosclerotic balloon-injured arteries by transfer of genes that inhibit cell proliferation. It is not known, however, whether vascular cell proliferation can be inhibited after injury in more extensively diseased atherosclerotic arteries. Accordingly, the purpose of this study was to investigate whether expression of recombinant genes in atherosclerotic arteries after balloon injury could inhibit intimal cell proliferation. To test this hypothesis, we examined the response to balloon injury in atherosclerotic rabbit arteries after gene transfer of herpesvirus thymidine kinase gene (tk) and administration of ganciclovir. Smooth muscle cells from hyperlipidemic rabbit arteries infected with adenoviral vectors encoding tk were sensitive to ganciclovir, and bystander killing was observed in vitro. In atherosclerotic arteries, a human placental alkaline phosphatase reporter gene was expressed in intimal and medial smooth muscle cells and macrophages, identifying these cells as targets for gene transfer. Expression of tk in balloon-injured hyperlipidemic rabbit arteries followed by ganciclovir treatment resulted in a 64% reduction in intimal cell proliferation 7 d after gene transfer (P = 0.004), and a 35-49% reduction in internal area 21 d after gene transfer, compared with five different control groups (P < 0.05). Replication of smooth muscle cells and macrophages was inhibited by tk expression and ganciclovir treatment. These findings indicate that transfer of a gene that inhibits cellular proliferation limits the intimal area in balloon-injured atherosclerotic arteries. Molecular approaches to the inhibition of cell proliferation in atherosclerotic arteries constitute a possible treatment for vascular proliferative diseases.

Role for tissue factor pathway in murine model of vascular remodeling.

Tissue factor (TF) is a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of arterial thrombogenicity. TF pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. The aim of this study was to define the complex interplay between TF and TFPI and the regulation of vascular thrombogenicity in a model of vascular remodeling. To determine the levels and pattern of vascular expression of TF and TFPI associated with vascular remodeling, a murine model of flow cessation was studied. TF activity of the arteries increased after ligation (P<0.05). Quantitative analysis of homogenates of remodeled carotid arteries revealed increased TF expression but unchanged TFPI expression compared with normal carotid arteries, resulting in enhanced TF activity. To determine the potential therapeutic role of TFPI in this thrombogenic state, mice were treated with intravascular adenoviral delivery of either murine TFPI (Ad-mTFPImyc) or a control adenovirus (Ad-DeltaE1). Overexpression of TFPI decreased vascular TF activity compared with viral control (P<0.01). Overexpression of TFPI inhibited neointimal formation (P=0.038), resulting in enhanced luminal area (P=0.001) 4 weeks after flow cessation. In this murine model of vascular remodeling, an imbalance between TF and TFPI expression is generated, resulting in increased TF activity. Overexpression of TFPI in this model inhibits vascular TF activity and results in attenuation of vascular remodeling associated with flow interruption.

Vascular actions of brain natriuretic peptide: modulation by atherosclerosis and neutral endopeptidase inhibition.

OBJECTIVES: We sought to define the vascular actions of the cardiac hormone brain natriuretic peptide (BNP) on cellular proliferation and cyclic guanosine monophosphate (cGMP) in human aortic vascular smooth muscle cells (HAVSMCs). Secondly, we investigated BNP and acetylcholine (ACh) vasorelaxations in aortic rings from normal and atherosclerotic rabbits in the presence and absence of long-term oral inhibition of neutral endopeptidase (NEP). BACKGROUND: The vascular actions of BNP are not well defined, despite the presence of its receptor in vascular smooth muscle and the upregulation of NEP, the ectoenzyme that degrades BNP, in the vascular wall in atherosclerosis. METHODS: HAVSMCs stimulated with fetal calf serum (FCS) were pulsed with bromodeoxyuridine (BrdU) with and without BNP. The HAVSMCs were incubated in the presence and absence of BNP to assess cGMP. Vasorelaxations to BNP and ACh were assessed in rings in normal and atherosclerotic rabbits in the presence and absence of long-term oral inhibition of NEP, together with assessment of atheroma formation. RESULTS: FCS-stimulated BrdU uptake in HAVSMCs was suppressed with BNP. BNP potentiated cGMP in HAVSMCs. BNP resulted in potent vasorelaxation in normal isolated aortic rings, which were impaired in atherosclerotic versus normal rabbits and preserved with NEP inhibition, which also decreased atheroma formation. Relaxations to ACh, which were also impaired in atherosclerosis, were preserved with inhibition of NEP. CONCLUSIONS: We conclude that BNP potently inhibits vascular smooth muscle cell proliferation and potentiates the generation of cGMP. BNP potently relaxes the normal rabbit aorta, and this response is impaired in atherosclerosis but preserved with inhibition of NEP, together with a reduction in atheroma formation and preservation of relaxations to ACh.

Simvastatin preserves coronary endothelial function in hypercholesterolemia in the absence of lipid lowering.

Recent evidence suggests that some benefit from the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors may occur independent of lipid lowering. We aimed to determine the effect of simvastatin on coronary endothelial function, endothelial NO synthase (eNOS) expression, and oxidative stress in experimental hypercholesterolemia (HC) in the absence of cholesterol lowering. Pigs were randomized to 3 experimental groups: normal diet (N group), high cholesterol diet (HC group), and HC diet with simvastatin (HC+S group) for 12 weeks. Low density lipoprotein cholesterol was similarly increased in the HC and HC+S groups compared with the N group. In vitro analysis of coronary large- and small-vessel endothelium-dependent vasorelaxation was performed. The mean vasorelaxation of epicardial vessels to bradykinin was significantly attenuated in the HC group compared with the N group (32.3+/-1.2% versus 42.9+/-1.6%, respectively; P<0.0001). This attenuation was significantly reversed in the HC+S group (38.7+/-1.5%, P<0.005 versus HC group). The maximal vasorelaxation to substance P was significantly attenuated in the HC group compared with the N group (50.5+/-11.9% versus 79.3+/-5.3%, respectively; P<0.05). This attenuated response was normalized in the HC+S group (74.9+/-4.1%, P<0.05 versus HC group). The maximal arteriolar vasorelaxation to bradykinin was also significantly attenuated in the HC group compared with the N group (71.9+/-4.9% versus 96.8+/-1.34%, respectively; P<0.005). This was reversed in the HC+S group (98.4+/-0.6%, P<0.0001 versus HC group). Western blotting of coronary tissue homogenates for eNOS demonstrated a decrease in protein levels in the HC group compared with the N group, with normalization in the HC+S group. Elevation of plasma F(2)-isoprostanes and thiobarbituric acid-reactive substances, markers of oxidative stress, occurred in the HC compared with the N group. These changes were reversed in the HC+S group. In summary, simvastatin preserves endothelial function in coronary epicardial vessels and arterioles in experimental HC (in the absence of cholesterol lowering) in association with an increase in coronary eNOS levels and a decrease in oxidative stress. These alterations may play a role in the reduction in cardiac events after treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

A new cationic liposome DNA complex enhances the efficiency of arterial gene transfer in vivo.

An important goal of gene therapy for cardiovascular diseases and cancer is the development of effective vectors for catheter-based gene delivery. Although adenoviral vectors have proven effective for this purpose in animal models, the ability to achieve comparable gene transfer with nonviral vectors would provide potentially desirable safety and toxicity features for clinical studies. In this report, we describe the use of a new cationic DNA-liposome complex using an improved expression vector and lipid, N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleyl phosphatidylethanolamine (GAP-DL-RIE/DOPE) to optimize catheter-mediated gene transfer in porcine arteries. The efficiency of this vector was compared to DNA alone, DNA with a previously described cationic liposome complex, (+/-)-N-(2-hydroxyethyl)-N, N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE/DOPE), and a replication-defective adenoviral vector in a porcine artery gene transfer model. When used in optimal ratios, GAP-DL-RIE/DOPE liposomes provided a 15-fold higher level of gene expression in arteries compared to DNA alone or DMRIE/DOPE. Gene expression was observed in intimal and medial cells. However, when compared to adenoviral vectors (10(10) pfu/ml), gene expression following GAP-DLRIE/DOPE transfection was approximately 20-fold lower. Following intravenous injection of GAP-DLRIE/DOPE in mice, biochemical, hematological, and histopathological abnormalities were not observed. Significant improvements in the efficacy of arterial gene expression can be achieved by optimization of transfection condition with DNA-liposome complexes in vivo that may prove useful for arterial gene delivery in cardiovascular diseases and cancer.

Autocrine role for the endothelin-B receptor in the secretion of adrenomedullin.

Adrenomedullin, originally discovered in human pheochromocytoma, is a vasodilating and natriuretic peptide of vascular endothelial and smooth muscle cell origin. Although endothelin-1 (ET-1) has been implicated as a vasoconstricting and growth-promoting peptide of endothelial origin, it may more importantly function as an autocrine factor and release vasodilatory substances such as nitric oxide by mechanisms linked to the endothelin-B (ETB) receptor subtype. The present study was designed to establish that the ETB receptor stimulates the secretion of adrenomedullin from cultured canine aortic endothelial cells. We first sought to determine the presence and production of adrenomedullin in canine aortic endothelial cells using immunohistochemistry and Northern blot analysis, which revealed that adrenomedullin immunoreactivity and adrenomedullin mRNA were present in canine aortic endothelial cells. Second, adrenomedullin was time-dependently secreted from canine aortic endothelial cells, with a secretion rate of 15.7+/-1.5 pg/10(5) cells per 24 hours. Furthermore, immunohistochemistry revealed the presence of the ETB receptor in canine aortic endothelial cells, and ETB receptor stimulation by sarafotoxin S6c increased adrenomedullin production and secretion from canine aortic endothelial cells. Such actions were blocked with the ETB receptor antagonist IRL-2500 but not with ETA receptor antagonist FR-139317. These studies are the first to report an additional autocrine role of the ETB receptor in the release of vasodilating and natriuretic peptide adrenomedullin, and they suggest another important vasoactive system regulated by the ET receptor subtype.

Activated nuclear factor-kappaB is present in the coronary vasculature in experimental hypercholesterolemia.

BACKGROUND: Experimental hypercholesterolemia (HC) is characterized by a decrease in nitric oxide (NO) bioavailability and cellular proliferation. Nuclear factor-kappaB (NF-kappaB) is a transcriptional factor which plays a coordinating role in inflammation and cellular proliferation and may be involved in early atherosclerosis. We examined whether activated NF-kappaB was present in experimental hypercholesterolemia in the coronary vasculature in association with a decrease in NO bioavailability. METHODS: A total of 14 juvenile domestic crossbred pigs were placed on a HC diet and six pigs on a normal diet for 10-12 weeks. A monoclonal antibody to the activated form of the p65 subunit of NF-kappaB was used to detect immunoreactivity in coronary artery sections. Coronary tissue homogenates were analyzed for activated NF-kappaB and endothelial nitric oxide synthase (eNOS) using Western blotting. In vitro coronary endothelium-dependent relaxation was performed in response to bradykinin, as a measure of NO bioavailability. RESULTS: Intimal staining for activated NF-kappaB was present in 12/14 HC pigs as compared with 0/6 controls (P<0.001). Confocal microscopy confirmed the presence of NF-kappaB in the nucleus of intimal cells although the majority of the staining was cytoplasmic. In the HC group, Western blotting revealed an increase in activated NF-kappaB in the vessel wall compared to the normal group, in association with a decrease in the presence of eNOS protein and an attenuated vasorelaxation response to bradykinin. CONCLUSION: This study suggests a potential role for activation of NF-kappaB, in association with a decrease in NO bioavailability, in the initial stages of atherosclerosis in the coronary vasculature.

Coronary artery apoptosis in experimental hypercholesterolemia.

The altered coronary vasoactivity detected in experimental hypercholesterolemia before lesion formation is presumably due to an imbalance between vasodilating and vasoconstricting factors. Apoptosis, which has been previously described in advanced atherosclerosis, is modulated by vascular derived peptides with vasoactive properties. We hypothesized that coronary apoptosis occurs in experimental hypercholesterolemia prior to lesion formation. Pigs were sacrificed after being on either a high-cholesterol diet for 10-16 weeks (n = 17) or a normal diet (n = 9). Identification of apoptosis in each layer of coronary arteries and arterioles was performed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL). In additional animals, ligation-mediated polymerase chain reaction (PCR) and transmission electron and confocal microscopy were done. Plasma cholesterol levels were higher in the cholesterol-fed animals (86+/-9 mg/dl versus 342+/-20 mg/dl, P < 0.001). Atheromatous plaques were not evident in the high-cholesterol group. TUNEL was positive in 11 of 17 hypercholesterolemic animals, primarily in the intima (1-2% of cells) and adventitia (3% of cells), but not in control vessels. A similar distribution was detected in arterioles. DNA bands were detected only in experimental animals, as were morphological features of apoptosis by transmission electron and confocal microscopy. In experimental hypercholesterolemia, apoptosis occurred in coronary arteries and arterioles before lesion formation. Apoptosis may be an integral process of early coronary atherosclerosis.

Plasmin proteolysis of endothelial cell and vessel wall associated tissue factor pathway inhibitor.

Plasmin is an important protease that mediates clot fibrinolysis and vessel wall extracellular matrix proteolysis. Recently, in vitro studies have suggested that plasmin can cleave and inactivate recombinant TFPI, a major inhibitor of TF-mediated coagulation. We hypothesized that such an interaction may occur in vascular cells expressing TFPI, or in the vessel wall, with implications for thrombolysis. In a series of experiments, we examined the effects of plasmin on cell surface and extracellular matrix (ECM) associated TFPI in endothelial cells (EC) in culture and on EC and smooth muscle cells (SMC) in the vessel wall. Plasmin (0.2 microM) decreased cell surface and matrix associated TFPI activity in cultured endothelial cells by 77 +/- 5% and 69 +/- 6% respectively (p < 0.01). Plasminogen, the proenzyme form of plasmin had no such effect on cell surface TFPI or matrix TFPI. Cell surface TFPI antigen measured by fluorescence activated cell sorter (FACS) was also significantly reduced by plasmin. Proteolysis of conditioned medium TFPI was suggested by loss of a approximately 45kD TFPI on Western Blot analysis following plasmin treatment. Plasmin also proteolysed a approximately 45kD TFPI protein in the intact ECM of EC, an effect which was inhibited by preincubation with aprotinin, a plasmin inhibitor. Incubation of similar concentrations of plasmin, with homogenates of normal vessel decreased a approximately 45kD TFPI immunoreactive band on Western blot analysis. Plasmin also decreased surface TFPI activity on frozen sections of normal vessel as measured by an amidolytic assay. Finally, plasmin treatment of atherosclerotic plaque sections caused complete removal of TFPI immunoreactivity associated with luminal EC and intimal SMC, when compared to control treated plaque (n = 3). Together these data suggest that plasmin proteolyses the majority of EC-associated (surface and matrix) TFPI and may remove TFPI from the luminal surface and intima of the vessel wall. TFPI proteolysis in cultured EC was associated with significant reduction in TFPI anticoagulant activity. These data provide evidence that plasmin degradation of TFPI occurs in vascular cells and in the vessel wall and may have implications for re-thrombosis following thrombolysis in vivo.

Capabilities of supine exercise electrocardiography versus exercise radionuclide angiography in predicting coronary events.

The ability of supine exercise electrocardiography and exercise radionuclide angiography to predict time to subsequent cardiac events (cardiac death, nonfatal myocardial infarction or late coronary bypass grafting or angioplasty) were compared in 265 patients with normal resting electrocardiograms who were not taking digoxin. All patients had undergone coronary catheterization and were initially treated medically. Follow-up study was performed at a median of 51 months. Separate logistic regression models, which had been previously developed to predict 3-vessel or left main coronary artery disease (CAD), were compared using a Cox regression analysis to predict time to a subsequent cardiac event. The exercise electrocardiography model, consisting of the magnitude of ST depression, exercise heart rate and patient gender, was a powerful predictor (chi-square = 30.8, p less than 0.0001) of subsequent events. The exercise radionuclide angiography model, which included the exercise response of the pressure-volume ratio in addition to the exercise electrocardiography variables, had similar prognostic power (chi-square = 31.8, p less than 0.0001). In a separate analysis considering only cardiac death and nonfatal myocardial infarction, the exercise electrocardiography model remained a significant predictor of events (chi-square = 12.2, p less than 0.001). None of the radionuclide angiography variables added significantly to the prognostic power of the exercise electrocardiography model. Thus, in patients with a normal resting electrocardiogram who are not taking digoxin, the supine exercise electrocardiography model that predicts 3-vessel or left main CAD also predicts future cardiac events. Exercise radionuclide angiography does not provide any additional prognostic information in such patients.

Gene therapy for atherosclerotic cardiovascular disease: a time for optimism and caution.

Cardiovascular disease is the leading cause of death in the Western world, and gene therapy approaches to several cardiovascular disorders have been proposed. One of the major stumbling blocks to be overcome before widespread clinical use of this technology is how to deliver DNA efficiently and safely to cells in vivo. While delivery of DNA alone is inefficient, use of viral vectors may overcome this problem. Adenoviral vectors are most commonly used in cardiovascular gene delivery, but toxicity related to these vectors remains a concern. In addition, duration of gene expression with use of these vectors is limited, which may be advantageous in settings in which transient expression is satisfactory to obtain a therapeutic effect. Gene therapy has been suggested as an approach to multiple conditions, including restenosis after angioplasty, therapeutic neovascularization, and bypass graft restenosis. Phase 1 clinical trials were recently reported. While proof of principle has been established in preclinical animal models, convincing efficacy data in humans do not yet exist. Improvements in vector technology and methods of catheter-mediated vascular gene delivery are needed before widespread clinical application of this therapy.

Percutaneous transluminal coronary angioplasty and the changing indications for coronary artery bypass grafting for single-vessel coronary artery disease.

Patients with single-vessel coronary artery disease have a good long-term prognosis with either medical or surgical therapy. Because percutaneous transluminal coronary angioplasty has become widely available for treating patients with symptomatic single-vessel coronary artery disease, those who currently undergo coronary artery bypass grafting may be a select group. In this study, we examined the effects of the increasing use of percutaneous transluminal coronary angioplasty on the indications for coronary artery bypass grafting in patients with symptomatic single-vessel coronary artery disease and reviewed the type of procedures performed in such patients at our institution between 1983 and 1988. During this period, 115 patients underwent coronary artery bypass grafting for single-vessel coronary artery disease. The indication for revascularization was angina in 111 patients (88% were in class III or IV, Canadian Cardiovascular Society classification), acute myocardial infarction in 3, and a strongly positive result of an exercise test in 1. The number of surgical revascularization procedures annually for single-vessel coronary artery disease remained consistent throughout the study period. In a comparison of the first 3 years of the study with the last 3 years, the number of patients who underwent coronary artery bypass grafting for restenosis after coronary angioplasty increased, but the number who had surgical revascularization because of failure of coronary angioplasty decreased. In addition, more patients received internal mammary grafts during the second half of the study (42 or 72%) than during the first half (24 or 42%).(ABSTRACT TRUNCATED AT 250 WORDS)

Coronary angioplasty in acute myocardial infarction: primary, immediate adjunctive, rescue, or deferred adjunctive approach?

OBJECTIVE: To address the current clinical applications, outcomes, and limitations of coronary angioplasty in the setting of acute myocardial infarction. DESIGN: We review the results of several large trials in which various strategies of thrombolysis and primary, immediate adjunctive, rescue, or deferred adjunctive coronary angioplasty were used in patients with acute myocardial infarction. MATERIAL AND METHODS: Four strategies for the utilization of angioplasty in myocardial infarction have been developed and are based on the timing and concurrent use of thrombolytic therapy. RESULTS: Primary coronary angioplasty without prior thrombolytic therapy is as effective as thrombolytic therapy for salvaging myocardium. Results of a meta-analysis of recent trials suggest potential benefits of increased survival and decreased reinfarction in comparison with the results of thrombolysis in recent trials. Immediate adjunctive angioplasty after thrombolytic therapy has been tested in three large, randomized trials. The results suggest that this strategy is associated with increased risks without benefits of increased survival or improved left ventricular function. Rescue angioplasty may be helpful after failed thrombolytic therapy. Ongoing randomized trials might further clarify the benefits of rescue angioplasty. Because of the inherent difficulty in the noninvasive identification of patients with persistent reocclusion, diagnostic coronary angiography early after thrombolytic therapy may be necessary. Deferred adjunctive angioplasty during the weeks after infarction to prevent recurrent ischemia was not shown to decrease mortality or reinfarction in two large trials. CONCLUSION: Primary coronary angioplasty is the treatment of choice for patients with contraindications to thrombolytic therapy. Certain high-risk subgroups may also benefit from primary angioplasty.

Nonviral vector-mediated thymidine kinase gene transfer and ganciclovir treatment in leiomyoma cells.

OBJECTIVE: To test the hypotheses that ganciclovir is cytotoxic to leiomyoma cells transfected with herpes simplex virus thymidine kinase and that estrogen modulates the responsiveness of tumor cells to this gene therapy approach. METHODS: Human and rat cultured uterine leiomyoma cells were transfected with plasmids encoding the beta-galactosidase gene, thymidine kinase gene, or a control plasmid. Transfection efficiency was monitored by measuring beta-galactosidase enzyme activity. Ganciclovir cytotoxicity in thymidine kinase-transfected cells was assessed by monitoring cell viability using trypan blue exclusion. The "bystander effect," a phenomenon in which thymidine kinase-expressing cells exposed to ganciclovir are toxic to adjacent thymidine kinase-nonexpressing cells, was assessed when thymidine kinase vector-transfected cells were cocultured with control plasmid-transfected cells at various percentages before exposure to ganciclovir. The effect of estradiol on ganciclovir-thymidine kinase-mediated cytotoxicity was assessed in estrogen-responsive rat leiomyoma cells. RESULTS: A thymidine kinase-ganciclovir-mediated "bystander effect" was demonstrated, with 48.6% (human) and 65.6% (rat) cell death when 5% of the leiomyoma cells were transfected with the pNGVL1-tk vector, with 0.84% and 1.9% of the cells expected to express thymidine kinase as based on the 16.7% and 39.8% transfection efficiency determined by the reporter gene assay in human and rat leiomyoma cells, respectively. Estradiol promoted cell growth and enhanced the "bystander effect" in rat leiomyoma cells. CONCLUSION: These findings demonstrate the feasibility of using thymidine kinase gene therapy as a novel treatment for uterine leiomyomas. The effect of estrogen may provide a mechanism to enhance the tumor-suppressive effect of this approach.

Clinical outcomes after successful percutaneous coronary angioplasty of saphenous vein graft disease and the importance of long-term assessment.

We examined the short- and long-term outcome of 327 patients at Mayo Clinic who had undergone coronary angioplasty of saphenous vein graft stenoses to determine whether the traditional 6-month assessment of clinical end points after coronary angioplasty is as useful as it is for patients who have had angioplasty of native vessel disease. Follow-up over 3.3+/-2.7 years was performed. At 6 months, 96+/-1% of the patients were alive, whereas at the 1- and 5-year end points, survival had deteriorated to 92+/-2 and 67+/-3%, respectively. Only 80+/-2% were free of severe angina at 6 months and 62+/-3 and 36+/-3% after 1 and 5 years. Combined event-free rate for death, Q-wave myocardial infarction or repeat coronary artery bypass surgery was 86+/-2% at 6 months, 78+/-2% at 1 year, and 45+/-4% at 5 years. Independent predictors of mortality included advancing age, diabetes mellitus, target vein grafts >5 years old and left ventricular ejection fraction <40%. In conclusion, despite reasonable 6- to 12-month outcomes following vein graft angioplasty, significant attrition in survival and event-free survival was observed. These observations have important implications for the interpretation of results of trials comparing conventional angioplasty and coronary bypass surgery with newer interventional devices if only the traditional 6-month follow-up interval is used.

A proliferation analysis of arterial neointimal hyperplasia: lessons for antiproliferative restenosis therapies.

Medial smooth muscle cell proliferation is frequently implicated as the major cause of coronary restenosis. Although antiproliferative agents have shown efficacy in animal studies, they are ineffective in human trials. To better understand these discrepancies, we performed a mathematical kinetic analysis of cellular proliferation in the neointimal hyperplasia of rats, pigs, and patients. A model was derived using a differential expression for proliferation, proportional to the number of cells present. Additional terms were included for inhibition of proliferation proportional to neointimal mass and time. The resulting equation was solved in closed form for the number of cells and proliferation rate. These equations were validated in the rat carotid artery injury model from published data. The model was then applied to the porcine coronary injury model, and then to clinical data obtained from angiographic human studies. Peak cellular proliferative activity in patients occurs at 16 days and continues at lower levels for much longer periods of time. Less than 10 generations of cells are sufficient to develop clinically significant restenosis. Conversely, proliferation rates in the two animal models (rats and pigs) are maximal at roughly 2 and 6 days, respectively, also continuing at low levels for extended time periods. Cell proliferation in restenosis is a highly controlled process, with comparatively few cell generations causing enough neointima for arterial obstruction to occur. Substantial cell kinetic differences occur across species. The rat exhibits high proliferation rates and rapid doubling times compared to patients and pigs, and is thus a highly 'proliferative' model. Such differences may be responsible for discrepant animal model and clinical trial results. These data may help determine the timing and strategy of therapy against clinical restenosis.

Enhanced angiogenesis and unfavorable remodeling in injured porcine coronary artery lesions: effects of local basic fibroblast growth factor delivery.

There is interest in the role of growth factors in the genesis of arterial remodeling. We studied local administration of basic fibroblast growth factor (bFGF) to coronary lesions to determine whether there is a difference in remodeling and whether neovascularization could be induced in such stenoses and distal myocardium. Pigs were randomized to balloon infusion of either saline or bFGF at each thermally injured arterial site. After the animals were killed, their internal elastic lamina, neointima, and lumen areas were measured. Capillaries were counted in the arteries and myocardium. There was a greater loss of lumen and internal elastic in the bFGF group. The neointima, media, and myocardium in the bFGF treated arteries had statistically more capillaries. This study showed that local intracoronary bFGF, at a dose that results in arterial luminal revascularization in injured segments, adversely affects arterial remodeling. Thus, the angiogenic response to exogenous bFGF may be offset by concomitant shrinkage of injured arterial segments.

Scaffolds for tissue engineering of cardiac valves.

Tissue engineered heart valves offer a promising alternative for the replacement of diseased heart valves avoiding the limitations faced with currently available bioprosthetic and mechanical heart valves. In the paradigm of tissue engineering, a three-dimensional platform - the so-called scaffold - is essential for cell proliferation, growth and differentiation, as well as the ultimate generation of a functional tissue. A foundation for success in heart valve tissue engineering is a recapitulation of the complex design and diverse mechanical properties of a native valve. This article reviews technological details of the scaffolds that have been applied to date in heart valve tissue engineering research.

Effect of enhanced external counterpulsation on circulating CD34+ progenitor cell subsets.

BACKGROUND: Enhanced external counterpulsation (EECP) is associated with improvement in endothelial function, angina and quality of life in patients with symptomatic coronary artery disease, although the mechanisms underlying the observed clinical benefits are not completely clear. The purpose of this study was to examine the effects of EECP on circulating haematopoietic progenitor cells (HPCs) and endothelial progenitor cells (EPCs) in patients with refractory angina. We compared HPC and EPC counts between patients scheduled for EECP and patients with normal angiographic coronary arteries, with and without coronary endothelial dysfunction. We hypothesized that an increase in circulating bone marrow derived progenitor cells in response to EECP may be part of the mechanism of action of EECP. METHODS: Thirteen consecutive patients scheduled to receive EECP treatment were prospectively enrolled. Clinical characteristics were recorded and venous blood (5 ml) was drawn on day 1, day 17, day 35 (final session) and one month post completion of EECP therapy. Buffy coat was extracted and HPCs and EPCs were counted by flow cytometry. RESULTS: Median Canadian Cardiovascular Society (CCS) angina class decreased and Duke Activity Status Index (DASI) functional score increased significantly (both, p < 0.05) in response to EECP, an effect that was maintained at one month after termination of treatment. Flow cytometric analysis revealed an accompanying significant increase in CD34+, CD133+ and CD34+, CD133+ CPC counts over the course of treatment (p < 0.05). DASI scores correlated significantly with CD34+ (R = 0.38 p = 0.02), CD133+ (R = 0.5, p = 0.006) and CD34+, CD133+ (R = 0.47, p = 0.01) CPC counts. CONCLUSION: This study shows that HPCs, but not EPCs are significantly increased in response to EECP treatment and correlate with reproducible measures of clinical improvement. These findings are the first to link the functional improvement observed with EECP treatment with increased circulating progenitor cells.