The "graphic experience" as clinical science: thoughts on the precordial leads of Wolferth and Wood.
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Coronary Disease↗
Biomedical subjects
M S Runge
Publications and source records attributed to M S Runge.
Oxidative stress and atherosclerosis: its relationship to growth factors, thrombus formation and therapeutic approaches.
The initiating event of atherogenesis is thought to be an injury to the vessel wall resulting in endothelial dysfunction. This is followed by key features of atherosclerotic plaque formation such as inflammatory responses, cell proliferation and remodeling of the vasculature, finally leading to vascular lesion formation, plaque rupture, thrombosis and tissue infarction. A causative relationship exists between these events and oxidative stress in the vessel wall. Besides leukocytes, vascular cells are a potent source of oxygen-derived free radicals. Oxidants exert mitogenic effects that are partially mediated through generation of growth factors. Mitogens, on the other hand, are potent stimulators of oxidant generation, indicating a putative self-perpetuating mechanism of atherogenesis. Oxidants influence the balance of the coagulation system towards platelet aggregation and thrombus formation. Therapeutic approaches by means of antioxidants are promising in both experimental and clinical designs. However, additional clinical trials are necessary to assess the role of antioxidants in cardiovascular disease.
Animals↗
Prevention of restenosis after percutaneous coronary interventions: the medical approach.
Restenosis following successful percutaneous coronary revascularization continues to represent a major problem limiting the clinical efficacy of this procedure. The underlying mechanisms of restenosis are comprised of a combination of effects from vessel recoil, negative vascular remodeling, thrombus formation and neointimal hyperplasia. Indeed, there are important interactions among all of these mechanisms. For example, neointimal hyperplasia is stimulated by growth factors, which are released by local thrombi and the injured arterial segment itself, and act to enhance the expression of other growth-regulating proteins, in particular "second messengers", proto-oncogenes and other cell cycle controlling proteins. This results in an inflammatory and myofibroproliferative response, which may worsen vessel narrowing caused by recoil and result in the formation of a clinically significant restenotic lesion. A multitude of pharmacologic trials have been conducted in an attempt to prevent restenosis, but most have demonstrated little benefit. Studies in smaller numbers of patients have suggested a potential benefit for several classes of agents, including: 1) the antiproliferatives, angiopeptin, trapidil and tranilast; 2) selective elimination or alteration of proliferating cells; 3) enhancement of natural growth inhibitors; and 4) signal transduction blockade or inhibition of the gene expression for various growth-stimulating proteins. Finally, there have been advances in related areas, including development of antithrombotic catheters, novel polymers, and more efficient methods for transferring genes into the vessel wall. All of these offer the possibility of delivering agents (drugs, genes, or antisense oligonucleotides) locally at the site of intervention in a way that may optimize antiproliferative effects while minimizing systemic effects--ultimately leading to a more specific inhibition of the restenosis process.
Angioplasty, Balloon, Coronary↗
Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells.
Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.
Aorta, Abdominal↗
Induction of rat aortic smooth muscle cell growth by the lipid peroxidation product 4-hydroxy-2-nonenal.
BACKGROUND: Atherosclerotic lesion formation is a complex process, in part mediated by inflammatory and oxidative mechanisms including lipid peroxidation. To further characterize the potential role of lipid peroxidation products in atherogenesis, we studied the effects of 4-hydroxy-2-nonenal (HNE) on rat aortic smooth muscle cell growth. METHODS AND RESULTS: HNE, at concentrations of 1.0 and 2.5 micromol/L, significantly stimulated rat aortic smooth muscle cell growth as determined by cell counts, [3H]-thymidine uptake, and incorporation of bromo-deoxyuridine. To characterize the mechanism of HNE-induced mitogenesis, its effect on activation of intracellular growth signaling pathways was examined. Treatment with HNE resulted in activation of extracellular signal-regulated protein kinases ERK1 and ERK2, induction of c-fos and c-jun protein expression, and an increase in transcription factor AP-1 DNA binding activity. In addition, HNE induced expression of platelet-derived growth factor-AA (PDGF-AA) protein, and an anti-PDGF-AA antibody specifically inhibited HNE-mediated DNA synthesis, suggesting that growth factor induction may play a role in HNE-induced vascular smooth muscle cell growth. The role of redox-sensitive mechanisms in this process was further supported by the observation that HNE-induced DNA synthesis and AP-1 activation were inhibited by the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate. CONCLUSIONS: These data demonstrate that HNE, one of several important lipid peroxidation products, induces rat aortic smooth muscle cell growth through redox-sensitive mechanisms and growth factor expression. These observations are consistent with a role for lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.
Aldehydes↗
Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells.
The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene expression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established that Sp1 and Sp3 both bind to this 3' GC box cluster. We mutated each of the three GC boxes individually and all three collectively within this 3' cluster. Basal promoter activity was decreased to 46%, 78% and 29% of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.
Aorta↗
Beta3 integrins are upregulated after vascular injury and modulate thrombospondin- and thrombin-induced proliferation of cultured smooth muscle cells.
BACKGROUND: Treatment with an antibody that binds beta3 integrins (abciximab; c7E3 Fab) at the time of coronary angioplasty decreases the need for repeat revascularization. Two potential mechanisms have been proposed to explain this effect: (1) inhibition of platelet aggregation or (2) interruption of ligand binding to beta3 integrins on the smooth muscle cell (SMC) surface. We examined the latter hypothesis by determining (1) if beta3 integrin expression is upregulated after vascular injury in the baboon, (2) if 7E3 binds beta3 integrins on cultured SMC, and (3) if beta3 integrin activation plays a role in proliferation of cultured SMC. METHODS AND RESULTS: Results demonstrated that immunostaining for beta3 integrins was present in the neointima 1 week after balloon withdrawal injury of baboon brachial arteries and that beta3 integrin expression colocalized with alpha-actin-positive cells. In contrast, staining for beta3 integrins was undetectable in contralateral uninjured brachial arteries. 7E3 bound to cultured human aortic SMC with an affinity (KD=3.3 nmol/L) similar to 7E3 binding to endothelial cells or platelets. Cotreatment with 7E3 partially inhibited thrombospondin-induced or alpha-thrombin-induced proliferation but not PDGF-induced or serum-induced proliferation. CONCLUSIONS: In summary, these studies demonstrate that vascular cell beta3 integrin expression is increased after injury, that 7E3 binds to cultured SMC with high affinity, and that beta3 activation is important for thrombospondin-induced or alpha-thrombin-induced proliferation. These results support the hypothesis that beta3 integrins play a role in SMC growth responses after balloon injury.
Abciximab↗
Arachidonic acid activates Jun N-terminal kinase in vascular smooth muscle cells.
We have previously demonstrated that arachidonic acid activates extracellular signal-regulated protein kinases (ERKs) group of mitogen-activated protein kinases (MAPKs) in vascular smooth muscle cells (VSMC). To understand the role of arachidonic acid in cellular signaling events, we have now studied its effect on jun N-terminal kinases (JNKs) group of MAPKs in VSMC. Arachidonic acid activated JNK1 in a time- and concentration-dependent manner with maximum effects at 10 min and 50 microM. Induced activation of JNK1 by arachidonic acid is specific as other fatty acids such as linoleic and stearic acids had no such effect. Indomethacin and nordihydroguaiaretic acid (NDGA), potent inhibitors of the cyclooxygenase (COX) and the lipoxygenase (LOX)/monooxygenase (MOX) pathways, respectively, had no effect on arachidonic acid activation of JNK1 suggesting that the observed phenomenon is independent of its metabolism through either pathway. However, 12-hydroperoxyeicosatetraenoic acid (12-HpETE), the LOX metabolite of arachidonic acid significantly induced JNK1 activity. Protein kinase C (PKC) depletion by prolonged treatment of VSMC with phorbol 12-myristate 13-acetate (PMA) resulted in partial decrease in the responsiveness of JNK1 to arachidonic acid suggesting a role for both PKC-dependent and -independent mechanisms in the activation of JNK1 by this important fatty acid. On the other hand, the responsiveness of JNK1 to 12-HpETE was completely abolished in PKC-depleted cells, suggesting a major role for PKC in 12-HpETE-induced JNK1 activation. IL-1beta and TNF-alpha activated JNK1 in a time-dependent manner with maximum effect at 10 min. Desensitization of JNK1 by arachidonic acid significantly reduced its responsiveness to both the cytokines. In addition, 4-bromophenacyl bromide (4-BPB), a potent and selective inhibitor of phospholipase A2 (PLA2), significantly attenuated the cytokine-induced activation of JNK1. Together, these results show that (1) arachidonic acid and its LOX metabolite, 12-HpETE, activate JNK1 in VSMC, (2) PKC-dependent and -independent mechanisms play a role in the activation of JNK1 by arachidonic acid and 12-HpETE, and (3) arachidonic acid mediates, at least partially, the cytokine-induced activation of JNK1.
Animals↗
Cardioprotection by local heating: improved myocardial salvage after ischemia and reperfusion.
BACKGROUND: Previous studies have shown that expression of the inducible 70-kD heat-shock protein (HSP72) by whole-body hyperthermia is associated with protection against ischemia-reperfusion injury. To develop techniques for regional elevation of heat-shock proteins that prevent extracardiac sequelae during whole-body hyperthermia, we sought to determine if local heating of the heart in vivo provides protection against ischemia-reperfusion injury in the rat. METHODS: A thermal probe was used to locally heat rat hearts at two adjacent sites on the epicardial surface of the left ventricle. Rats were subjected to either 30 minutes of sham surgery (control; n = 10) or two local applications of the probe at 42.5 degrees to 43.5 degrees C for 15 minutes each (n = 9). After 4 hours, rats were subjected to 30 minutes of regional ischemia followed by 120 minutes of reperfusion. Hearts were removed and area at risk and infarct area were determined. RESULTS: Localized heat stress resulted in a significant limitation of infarct size in heat-treated animals versus controls (mean +/- standard error of the mean infarct area/area at risk = 4.3% +/- 0.85 versus 19.2% +/- 3.4%; p < 0.005). Western blot experiments confirmed elevated HSP72 expression in left (heated) and right (nonheated) ventricular samples from treated animals (n = 6; left ventricular = 5.5-fold; right ventricular = 3.7-fold) compared with sham-operated controls. Controls treated with the probe at 37 degrees C (n = 4) showed no increases in HSP72. CONCLUSIONS: Local heating of the heart is associated with elevated levels of HSP72 and improved myocardial salvage. The increase in expression of HSP72 is not limited to the heated region, but extends into nonheated regions of the heart as well. This may lead to the development of new techniques that improve methods of myocardial revascularization and heart transplantation procedures.
Analysis of Variance↗
The role of secondary growth factor production in thrombin-induced proliferation of vascular smooth muscle cells.
Alpha-thrombin is a multifunctional serine proteinase that is concentrated at sites of vascular injury and has been implicated in vascular healing responses following balloon injury. In addition to its well-known hemostatic effects, thrombin stimulates smooth muscle cell (SMC) proliferation via binding of protease activated receptor-1 (PAR-1), a seven transmembrane, G-protein-coupled cell surface receptor. Following activation of this receptor, SMC produce and secrete various autocrine growth factors, including platelet-derived growth factor-AA (PDGF-AA), basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor (HBEGF), and transforming growth factor-beta (TGFbeta). The role that autocrine growth factors play in alpha-thrombin-induced proliferation will be discussed in this review.
Angina, Unstable↗
Perspectives on coronary stenting.
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Aged↗
High-voltage and high-salt buffer facilitates electroporation of human aortic smooth-muscle cells.
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Aorta↗
Nuclear protein interactions with the human KDR/flk-1 promoter in vivo. Regulation of Sp1 binding is associated with cell type-specific expression.
The endothelial cell type-specific tyrosine kinase KDR/flk-1 is a receptor for vascular endothelial growth factor and a critical regulator of endothelial cell growth and development. To study mechanisms of endothelial cell differentiation and gene regulation, we have analyzed the topology of the proximal promoter of human KDR/flk-1. A protected sequence between base pairs -110 and -25 was defined by in vitro DNase I footprinting analysis in human umbilical vein endothelial cells (HUVECs). Purified Sp1 alone produced similar protection, and electrophoretic mobility shift assays demonstrated that Sp1 was indeed the major nuclear protein binding to this region. Despite the cell type specificity of KDR/flk-1 expression, no cell type differences were observed in DNA-protein interactions in vitro. In contrast, in vivo footprinting assays demonstrated marked differences in core promoter interactions between cell types. Protection of Sp1 binding sites was observed in HUVECs by in vivo DNase I footprinting, whereas in human fibroblasts and HeLa cells a pattern consistent with nucleosomal positioning was observed. In vivo dimethylsulfate footprinting confirmed that DNA-protein interactions occurred within Sp1 elements in HUVECs but not in nonendothelial cells. It is possible that distant elements coordinate Sp1 binding and chromatin structure to regulate cell type-specific expression of KDR/flk-1.
Base Sequence↗
Antithrombotic potency of hirudin is increased in nonhuman primates by fibrin targeting.
BACKGROUND: Inhibition of thrombin by either the indirect thrombin inhibitor heparin or by more potent direct thrombin inhibitors such as hirudin reduces thrombus formation after arterial injury. The present study was designed to determine if a fibrin-specific thrombin inhibitor could, by local thrombin inhibition, prevent thrombosis more effectively. METHODS AND RESULTS: We first studied antithrombotic potency in vitro, comparing fibrin-targeted hirudin (recombinant hirudin covalently linked to the Fab' fragment of the anti-fibrin monoclonal antibody 59D8) to recombinant hirudin in baboon plasma. Fibrin-targeted hirudin was nine times more effective than recombinant hirudin in inhibiting fibrin deposition on experimental clot surfaces in baboon plasma (P < .01). The potency of fibrin-targeted hirudin was then compared with that of recombinant hirudin in a baboon model of thrombus formation. 111In-labeled platelet deposition was measured in a synthetic graft segment of an extracorporeal arteriovenous shunt in control animals and in animals receiving either fibrin-targeted hirudin or hirudin. In these experiments, fibrin-targeted hirudin was 10-fold more potent than hirudin in inhibiting platelet deposition and thrombus formation (P < .05). CONCLUSIONS: These data indicate that targeting a thrombin inhibitors such as hirudin to an epitope present in thrombi results in increased antithrombotic potency.
Animals↗
Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells.
The inducible cyclooxygenase, COX-2, has been associated with vascular inflammation and cellular proliferation. We have discovered that hypoxia increases expression of the COX-2 gene in human vascular endothelial cells in culture independent of other stimuli. Western analysis of human umbilical vein endothelial cells (HUVEC) revealed a greater than 4-fold induction of protein by hypoxia (1% O2). The steady-state level of COX-2 mRNA was correspondingly elevated by both Northern blot and reverse transcriptase-polymerase chain reaction analysis. Using electrophoretic mobility shift assays with antibody supershifting, we also found that hypoxia causes increased binding of NF-kappaB p65 (Rel A) to the one out of the two NF-kappaB consensus elements in the COX-2 promoter which is closest to the transcription start site of the COX-2 gene. Transfection of an immortalized human microvascular endothelial cell line (HMEC-1) with mutation reporter gene constructs and HUVEC with both mutation and deletion reporter gene constructs suggested that transcription of the COX-2 gene was enhanced by hypoxia. In transcription factor decoy experiments, hypoxic HUVEC were exposed in culture to 20 microM of the same NF-kappaB element found to bind NF-kappaB protein. The wild type transcription factor decoy prevented hypoxic induction of COX-2, presumably by binding with cytoplasmic p65; however, mutated or scrambled oligonucleotides did not prevent the increase in COX-2 protein expression by hypoxia. Thus, the intracellular signaling mechanism that leads to induction of COX-2 by hypoxia includes binding of p65 to the relatively 3' NF-kappaB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.
Base Sequence↗
Endothelial cells exhibit differential chemokinetic and mitogenic responsiveness to alpha-thrombin.
Rapid regeneration of the endothelium is a critical component of vascular wall repair because limitations of this process enhance early thrombotic and vasospastic complications, as well as late sequelae of recurrent lesion formation. We have postulated that direct activation of the thrombin receptor initiates both mitogenic and chemokinetic endothelial behavior which facilitates intimal repair. To characterize the role of the thrombin receptor in human endothelial cell (EC) proliferation and migration, we investigated the effects of both alpha-thrombin (0.5-10 U/ml) and its receptor-activating peptide (TRAP; 1-100 microM). Responses of human aortic (HAEC) and umbilical vein (HUVEC) were characterized using [3H]thymidine and 61Cr microcarrier bead assays of proliferation and migration, respectively. Expression of motility-related genes was evaluated using a ribonuclease protection assay. Thrombin exerts both of its chemokinetic and mitogenic effects differentially in human endothelial cells. Following 2 or 4 days in culture, HUVEC proliferation increased two- to threefold after exposure to thrombin, primarily in the low concentration range (P < 0.05). However, HACE proliferation was inhibited up to 50% after a 4-day incubation period (P < 0.005). These mitogenic effects, including the inhibition of aortic endothelial cell proliferation, were reproduced, in part, by thrombin receptor activation with TRAP. In contrast, thrombin stimulates migratory responses in HAEC, but not HUVEC. However, this behavior was not reproduced by TRAP. It is noteworthy that urokinase-plasminogen activator (u-PA) expression was much more strongly expressed in migrating HAEC than in the HUVEC population. Moreover, when stimulated with thrombin, u-PA gene expression was significantly augmented in HAEC. It has been speculated that an effective human thrombin receptor (HTR) antagonist may reduce the proliferation of vascular smooth muscle cells and the development of a restenotic lesion following arterial wall injury. Our data suggest that such an inhibitor will likely also accelerate intimal regeneration through a dominant effect on limiting the HTR inhibitory effect on endothelial proliferation.
Amino Acid Sequence↗
Vascular thrombin receptor regulation in hypertensive rats.
Thrombin has been implicated as an important mediator of vascular lesion formation in atherosclerosis and restenosis. To investigate a potential role for thrombin signaling in the vascular response to hypertension, we have studied thrombin receptor (TR) expression and regulation in hypertensive rats. Aortic TR mRNA was upregulated by angiotensin II (Ang II)-induced hypertension (10.7 +/- 2.5 times control, P < .02), which correlated with a 4-fold increase in thrombin-induced constriction in isolated endothelium-denuded aortic rings. The AT1 receptor antagonist losartan normalized blood pressure and TR mRNA. Conversely, lowering blood pressure to the same degree with hydralazine did not abolish the upregulation of TR mRNA expression. When low-renin low-Ang II hypertension was induced in Dahl salt-sensitive rats, there was no detectable increase in the expression of aortic thrombin receptor mRNA. Finally, treatment with a chimeric heparin-binding form of the recombinant human Cu/Zn superoxide dismutase caused complete inhibition of TR mRNA upregulation, suggesting that an increased rate of superoxide anion production is an important signaling mechanism. Thus, increased TR expression via a redox-sensitive mechanism in the aortic smooth muscle of rats treated with Ang II represents a novel in vivo mechanism through which the hypertensive effects of Ang II are mediated.
Angiotensin II↗
Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis.
Neovascularization is a hallmark of neointimal formation in atherosclerotic plaques and restenotic lesions. Vascular endothelial growth factor (VEGF) promotes neovascular growth, whereas oxidative stress is a potent factor in vascular cell proliferation. To investigate the mechanisms of neovascular formation, we treated human and rat vascular smooth muscle cells (VSMCs) with H2O2. Northern blot analysis demonstrated a dose- and time-dependent increase in VEGF mRNA, with a maximum of 4-fold at 3 hours (200 mumol/L). As determined by immunoblotting and enzyme-linked immunosorbent assay, VEGF protein expression and secretion were similarly increased. Human umbilical vein endothelial cells were treated with conditioned medium from VSMCs incubated with 200 mumol/L H2O2. DNA synthesis, measured by thymidine incorporation, was increased 4-fold compared with control, an effect that was blocked by a neutralizing anti-VEGF antibody. The lipid peroxidation product 4-hydroxynonenal (1 mumol/L), an endogenous reactive oxygen species present in human atherosclerotic lesions, also increased VEGF secretion in VSMCs in a similar time-dependent fashion. Immunohistochemical staining and in situ hybridization of aortic sections from balloon-injured baboons demonstrated increased VEGF expression in discrete areas of the neointima and media compared with control sections, and expression correlated with the generation of 4-hydroxynonenal. Regulators of VEGF expression, such as reactive oxygen species, may enhance neovascularization of atherosclerotic and restenotic arteries.
Animals↗