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Biomedical subjects

J M Isner

Publications and source records attributed to J M Isner.

At least 109 records · Page 6Linked to original sources
PubMed1997

Estrogen-receptor-mediated inhibition of human endothelial cell apoptosis. Estradiol as a survival factor.

BACKGROUND: A series of studies was performed to examine the ability of estradiol (E2) to protect endothelial cells from apoptosis. METHODS AND RESULTS: Light and transmission electron microscopy demonstrated typical features of apoptosis in human umbilical vein endothelial cells (HUVEC) exposed to tumor necrosis factor-alpha (TNF-alpha). Northern and Western blot analyses revealed induction of message and protein for the interleukin-1 beta converting enzyme (ICE), which has been shown to mediate apoptosis induced by TNF-alpha. Immunofluorescent staining of HUVEC colocalized ICE expression to apoptotic HUVEC. Direct cell counting demonstrated a significant decrease in total endothelial cell number after 24 hours of TNF-alpha exposure and a dose-dependent reversal of the effect of TNF-alpha with E2 treatment. This protective effect was abrogated by an estrogen-receptor antagonist. Fluorescence-activated cell sorting analysis revealed 39.3% apoptosis after 24 hours of TNF-alpha exposure. Treatment with E2 resulted in a 50% decrease in apoptosis. Similarly, viability assays revealed 35 +/- 4% cell death after TNF-alpha exposure. Simultaneous treatment with E2 resulted in a dose-dependent reduction of cell death to a minimum of 18 +/- 2%. The protective effect of E2 was nullified by a specific estrogen-receptor antagonist. CONCLUSIONS: E2 treatment resulted in a dose-dependent, receptor-mediated inhibition of TNF-alpha-induced endothelial cell apoptosis. These studies indicate that E2 may also serve a maintenance function in preventing endothelial cell death after noxious stimuli and suggest that the ICE pathway may mediate cytokine-induced apoptosis in endothelial cells. Preservation of endothelial integrity represents another mechanism that may account for the atheroprotective effect of estrogen.

Apoptosis↗
PubMed1997

Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium.

BACKGROUND: Vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) is an endothelial cell (EC) mitogen. This feature is considered central to the documented role of VEGF/VPF in promoting angiogenesis. More recent evidence suggests that VEGF/VPF may also serve a "maintenance" function, modulating various aspects of EC biology. In the present study, we sought to determine the extent to which VEGF/VPF may stimulate the release of NO from normal ECs. METHODS AND RESULTS: VEGF/VPF produced a dose-dependent rise in NO concentration ([NO]) from vascular segments of rabbit thoracic aorta, pulmonary artery, and inferior vena cava. In comparison to stimulation with acetylcholine, the onset of increased [NO] after administration of VEGF/VPF was slower, reaching a maximum value after 8 minutes. Preincubation of the aortic segments with L-arginine raised by twofold both baseline [NO] and [NO] stimulated by addition of 2.5 micrograms/mL VEGF/VPF. Removal of CaCl2 from the Krebs solution, disruption of the endothelium, and administration of NG-monomethyl-L-arginine abrogated the stimulatory effect of 10 micrograms/mL VEGF/VPF. Similar findings were documented with an NO-specific polarographic electrode to measure NO released from cultured human umbilical vein ECs. CONCLUSIONS: VEGF/VPF stimulates production of NO from rabbit and human ECs. This finding (1) constitutes inferential evidence for the presence of functional VEGF/VPF receptors on quiescent endothelium of the adult rabbit as well as human ECs and (2) supports the notion that putative maintenance functions of VEGF/VPF may include regulation of baseline synthesis and/or release of EC NO.

Acetylcholine↗
PubMed1997

Isolation of putative progenitor endothelial cells for angiogenesis.

Putative endothelial cell (EC) progenitors or angioblasts were isolated from human peripheral blood by magnetic bead selection on the basis of cell surface antigen expression. In vitro, these cells differentiated into ECs. In animal models of ischemia, heterologous, homologous, and autologous EC progenitors incorporated into sites of active angiogenesis. These findings suggest that EC progenitors may be useful for augmenting collateral vessel growth to ischemic tissues (therapeutic angiogenesis) and for delivering anti- or pro-angiogenic agents, respectively, to sites of pathologic or utilitarian angiogenesis.

Animals↗
PubMed1997

Stent endothelialization. Time course, impact of local catheter delivery, feasibility of recombinant protein administration, and response to cytokine expedition.

BACKGROUND: Because prior studies have established the critical role of the endothelium in preventing vascular thrombosis and intimal thickening, we designed a series of experiments to determine the feasibility of percutaneous local catheter delivery of recombinant protein to accelerate development of an intact endothelial monolayer after stent implantation. METHODS AND RESULTS: Balloon injury followed by percutaneous delivery of a 15-mm-long, balloon-expandable metallic stent was performed in 64 rabbit external iliac arteries (baseline diameter, 2.67 +/- 0.07 mm). Planimetric time-course analysis disclosed < 20% stent endothelialization at 4 days, < 40% at 7 days, and near-complete endothelialization at 28 days. The reporter protein horseradish peroxidase and the endothelial cell-specific recombinant protein vascular endothelial growth factor (VEGF) were each effectively delivered from a local delivery catheter (channel balloon catheter, ChB) after stent implantation. Although local catheter delivery (of vehicle control) itself mildly retarded the extent of stent endothelialization (10.6 +/- 2.9%) versus no local delivery (25.5 +/- 6.6%, P = .045), local ChB delivery of 100 micrograms VEGF overcame this catheter effect: By day 7, stent endothelialization was nearly complete (91.8 +/- 3.8%) (P < .0001 versus no local delivery). Consequently, stent thrombus was reduced in the VEGF-treated group (mural thrombus, 5.3 +/- 3.7%) versus no local delivery (29.3 +/- 6.8%, P = .006). Occlusive thrombus was seen only in the absence of local VEGF administration. CONCLUSIONS: (1) Local delivery of recombinant protein to the arterial wall is feasible after stent implantation, and (2) local delivery of the endothelial cell mitogen VEGF accelerates stent endothelialization, reducing stent thrombosis. These results thus establish a novel means by which the safety and/or bioactivity of endovascular stents may be further enhanced.

Angiography↗
PubMed1997

Vascular endothelial growth factor inhibits endothelial cell apoptosis induced by tumor necrosis factor-alpha: balance between growth and death signals.

A series of experiments was performed to determine whether vascular endothelial growth factor (VEGF), in addition to its endothelial cell specific mitogenic activity, can also protect endothelial cells from toxin-induced programmed cell death. Apoptosis was induced in endothelial cell culture with tumor necrosis factor-alpha (TNF-alpha). Simultaneous exposure of endothelial cells to VEGF resulted in a dose dependent inhibition of apoptosis when evaluated by: (1) direct counting of cells with morphologic features of apoptosis after acridine orange staining; (2) analysis of DNA fragmentation by (a) agarose gel electrophoresis and (b) fluorescence activated cell sorting (FACS); and (3) viability assays dependent upon mitochondrial function. Induction of fibronectin and beta 3 integrin expression in endothelial cells by VEGF suggests that altered adhesion molecule expression may explain this survival effect.

Animals↗
PubMed1997

Vascular endothelial growth factor attenuates myocardial ischemia-reperfusion injury.

BACKGROUND: Hypoxic endothelial cell activation plays a key role in the myocardial dysfunction resulting from ischemia-reperfusion injury. Recent evidence suggests that vascular endothelial growth factor (VEGF) may, in addition to promoting angiogenesis, modulate various aspects of endothelial function and repair. We examined whether administration of VEGF in the cardioplegic solution might have a beneficial effect on myocardial ischemia-reperfusion injury in an isolated rat heart model. METHODS: Hearts from Sprague-Dawley rats were perfused with Krebs-Henseleit solution in a modified Langendorff apparatus. Percent recovery of cardiac output, coronary flow, stroke work, and percent increase in coronary vascular resistance were measured after 2 hours of global ischemia and 40 minutes of reperfusion. Coronary effluent was collected after ischemia and reperfusion for measurement of creatine kinase. RESULTS: Hearts receiving cardioplegia solution containing 125 microg VEGF showed significantly improved recovery of cardiac output, coronary flow, and stroke work, and significantly reduced coronary vascular resistance compared with hearts receiving hyperkalemic cardioplegia only (p < 0.05). Coadministration of a nitric oxide synthase inhibitor attenuated the VEGF-induced cardiprotective effects. Hearts treated with VEGF released significantly less creatine kinase compared with control hearts. CONCLUSIONS: Addition of VEGF to hyperkalemic cardioplegia protects against myocardial ischemia-reperfusion injury in the isolated rat heart.

Animals↗
PubMed1997

Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty.

OBJECTIVES: The expression of gax, an anti-proliferative homeobox gene, is rapidly downregulated in vascular smooth muscle cells (VSMCs) following arterial injury. Here we performed percutaneous adenovirus-mediated gene transfer into the iliac arteries of normal rabbits using a channel balloon catheter to assess the effects of gax overexpression on neointima formation, lumen diameter, reendothelialization and functional vasomotion. METHODS: A channel balloon catheter was used to perform both the arterial injury and local gene delivery. In each animal both iliac arteries were randomly assigned to receive either an adenovirus expressing the gax gene (Ad-Gax) or the beta-galactosidase gene (Ad-beta gal). In a second group of animals arteries were randomly assigned to receive either Ad-beta gal or saline. RESULTS: At one month, angiography revealed 36% less luminal narrowing in the Ad-Gax-treated arteries relative to the Ad-beta gal-treated control arteries. Histological analysis revealed that the intimal/medial ratio (I/M) was reduced by 56% in the Ad-Gax group. Endothelium-dependent vasomotion was not affected by the gax gene transfer. In the second group, no statistically significant differences were found between the saline and the Ad-beta gal-treated vessels for any of these parameters. CONCLUSIONS: Percutaneous adenovirus delivery of the gax gene to rabbit iliac arteries following endothelial denudation and vessel wall injury reduces neointimal hyperplasia and luminal stenosis, but does not affect endothelium-dependent vasomotion. This study demonstrates that a VSMC transcription factor can potentially be utilized for the development of a molecular therapy for vascular disorders.

Adenoviridae↗
PubMed1997

Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA.

OBJECTIVE: Previous studies have demonstrated that arterial gene transfer of naked DNA encoding for a secreted protein may permit modulation of the host phenotype despite a low transfection efficiency. Acidic fibroblast growth factor (aFGF) is an angiogenic growth factor, but is not secreted by intact cells. In the current study, we investigated the hypothesis that addition of a hydrophobic leader sequence to achieve active secretion of the gene product would permit therapeutic angiogenesis following arterial gene transfer of naked DNA encoding for aFGF. METHODS: Ten days following surgical induction of unilateral hindlimb ischemia, New Zealand white rabbits were randomized to intra-arterial gene transfer with one of three plasmids: p267 (encoding non-secreted aFGF, n = 10), pMJ35 (encoding secreted aFGF) (n = 10), or 500 micrograms of pGSVLacZ (control, n = 10) (500 micrograms each). All animals were studied at 30 days post-gene transfer for evidence of therapeutic angiogenesis. RESULTS: pMJ35 transfectants had more angiographically visible collaterals (angiographic score = 0.76 +/- 0.02) than either p267 (0.55 +/- 0.02, p < 0.01) or LacZ (0.47 +/- 0.02, p < 0.001). Limb blood pressure ratio for pMJ35 was 0.88 +/- 0.02 vs. 0.68 +/- 0.04 for p267 (p < 0.01) and 0.57 +/- 0.04 for LacZ (p < 0.001). Vascular resistance was significantly lower in the pMJ35 group, compared with that in pGSVLacZ group, both in resting state (3.2 +/- 0.4 vs. 7.4 +/- 1.4 respectively, p < 0.05) and after the administration of nitroprusside. Capillary density (per mm2) was also superior in pMJ35 group (274 +/- 10) vs. p267 (204 +/- 9, p < 0.01) and LacZ (177 +/- 6, p < 0.001). CONCLUSION: The paracrine effects of a secreted gene product may obviate the need for adjunctive vectors in strategies of arterial gene therapy.

Analysis of Variance↗
PubMed1997

Differential expression of nonmuscle myosin II isoforms in human atherosclerotic plaque.

Intimal proliferation and functional changes involving vascular smooth muscle cells are key events in the development of atherosclerosis, including restenosis after percutaneous transluminal angioplasty. Nonmuscle myosin (NMM) is required for cytokinesis and has been shown in cultures of vascular smooth muscle cells to undergo changes of isoform expression depending on the stage of proliferation and differentiation. The purpose of this study was to examine the differential expression of the two most recently identified nonmuscle myosin heavy chain isoform II (NMMHC-II) isoforms A and B in atherosclerotic plaque. Primary atherosclerotic and restenotic atherectomy specimens and non-atherosclerotic controls, were analyzed by Western Blot analysis, immunohistochemistry and in situ hybridization. Nonmuscle myosin heavy chain isoform IIA (NMMHC-IIA) was equally expressed in all types of tissue specimens both at the protein and mRNA levels. In contrast, NMMHC-IIB protein was found in restenotic specimens and normal artery but was at very low levels in primary atherosclerotic plaque. By in situ hybridization NMMHC-IIB mRNA levels were significantly greater in restenotic versus primary atherosclerotic lesions. NMMHC-IIB expression is associated with vascular restenosis but is downregulated in stable atherosclerotic lesions, whereas NMMHC-IIA is expressed in both. These results indicate that these new myosin isoforms have different functions and should be regarded separately with respect to smooth muscle proliferation and restenosis. They should prove to be useful molecular markers for the study of atherosclerosis and restenosis.

Aged↗
PubMed1997

Restenosis of endovascular stents from stent compression.

OBJECTIVES: We sought to determine the basis for restenosis within superficial femoral arteries (SFAs) and hemodialysis conduits treated with balloon-expandable stents. BACKGROUND: Use of stents within coronary and peripheral vessels continues to increase exponentially. The mechanism of restenosis within stents placed at various vascular sites is not well understood. In particular, the implications of deploying a balloon-expandable stent in a compressible site are not well understood. METHODS: After the serendipitous detection of stent deformation during intravascular ultrasound (IVUS) examination of a restenosed dialysis fistula, we evaluated a consecutive series of patients with stents placed in compressible vascular sites, including the SFA (six patients) and hemodialysis fistulae (five patients). Clinical, angiographic and IVUS examinations were performed to evaluate mechanisms of restenosis. RESULTS: Stent compression was identified as the principal cause of restenosis in all dialysis conduits and SFAs. Stent deformity was not reliably identified by angiography; however, IVUS identified compression of two forms: eccentric deformation, implicating two-point compressive force, and complete circumferential encroachment of stent struts around the catheter, suggesting multidirectional compressive force. Despite redilation, secondary restenosis resulting from recurrent compression recurred in most sites. CONCLUSIONS: Restenosis within balloon-expandable endovascular stents may occur as a result of stent compression, a phenomenon readily detected by IVUS, but often not by angiography. These findings have significant implications for the use of balloon-expandable stents within vascular sites subject to extrinsic compression, such as hemodialysis conduits, the adductor canal segment of the SFA and carotid arteries.

Aged↗
PubMed1997

Passivation of metallic stents after arterial gene transfer of phVEGF165 inhibits thrombus formation and intimal thickening.

OBJECTIVES: This study sought to test the hypothesis that direct gene transfer of an endothelial cell mitogen could passivate metallic stents by accelerating endothelialization of the prosthesis. BACKGROUND: Thrombosis and restenosis comprise the principal clinical manifestations of compromised biocompatibility of endovascular stents. Previous studies have demonstrated that endothelial recovery at sites of balloon injury is a critical determinant of consequent intimal thickening and mural thrombus. We therefore investigated the potential for an endothelial cell mitogen delivered as plasmid DNA to optimize stent biocompatibility. METHODS: Naked plasmid DNA encoding vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) (phVEGF165) was delivered locally using a hydrogel-coated balloon angioplasty catheter to 16 rabbit iliac arteries in which metallic stents had been placed at the site of balloon injury; the contralateral iliac artery of each rabbit was balloon injured and stented but not transfected. RESULTS: Stent endothelialization was accelerated by phVEGF165 gene transfer (87.38 +/- 5.06% vs. 33.13 +/- 9.73% [mean +/- SEM] of the planimetered stent surface in the treated vs. contralateral limb, p = 0.005). This was associated with a significant reduction in mural thrombus (3.7 +/- 2.4% vs. 32.7 +/- 9.7%, p = 0.01) at day 7 and intimal thickening (maximal intimal area 0.61 +/- 0.09 vs. 1.44 +/- 0.12 mm2, p < 0.0001) at day 28. No benefit was observed from pCMV-luciferase in 14 similarly instrumented control rabbits. CONCLUSIONS: These findings indicate that arterial gene transfer of naked plasmid DNA encoding for an endothelial cell mitogen may successfully passivate endovascular stents by accelerating stent endothelialization, thereby reducing in-stent thrombus and obstruction due to intimal thickening.

Angioplasty, Balloon↗
PubMed1997

Reciprocal relation between VEGF and NO in the regulation of endothelial integrity.

Balloon angioplasty disrupts the protective endothelial lining of the arterial wall, rendering arteries susceptible to thrombosis and intimal thickening. We show here that vascular endothelial growth factor (VEGF), an endothelial cell mitogen, is upregulated in medial smooth muscle cells of the arterial wall in response to balloon injury. Both protein kinase C (PKC) and tyrosine kinase pp60src mediate augmented VEGF expression. In contrast, nitric oxide (NO) donors inhibit PKC-induced VEGF upregulation by interfering with binding of the transcription factor activator protein-1 (AP-1) to the VEGF promoter. Inhibition of VEGF promoter activation suggests that NO secreted by a restored endothelium functions as the negative feedback mechanism that downregulates VEGF expression to basal levels. Administration of a neutralizing VEGF antibody impaired reendothelialization following balloon injury performed in vivo. These findings establish a reciprocal relation between VEGF and NO in the endogenous regulation of endothelial integrity following arterial injury.

Animals↗
PubMed1997

Improved efficiency of arterial gene transfer by use of poloxamer 407 as a vehicle for adenoviral vectors.

Improvement in the efficiency of adenovirus-mediated arterial gene transfer may augment the utility of cardiovascular gene therapy. In vitro studies suggest that poloxamer 407 enhances transfection efficiency of adenoviral vectors in vascular smooth muscle cells. The aim of the present study was to investigate whether poloxamer 407 facilitates adenovirus-mediated arterial transfection in vivo as well. Gene transfer was performed in balloon-injured rat carotid arteries using E1- adenoviral vectors diluted in either poloxamer 407 or phosphate buffered saline (PBS). Transfection efficiency was significantly higher in rats transfected using a nuclear beta-galactosidase expressing adenovector diluted in poloxamer 407 versus PBS (morphometry, 13.2 +/- 1.3% versus 4.1 +/- 0.4% transfected medial cells, P = 0.0001; chemiluminescence; 1.4 +/- 0.2 versus 0.4 +/- 0.2 mU beta-galactosidase/mg protein, P = 0.004). Moreover, in the presence of poloxamer 407, it was possible to reduce the incubation time of adenoviral vectors from 20 to 10 min without compromising transfection efficiency. Poloxamer 407 did not evoke specific tissue toxicity. Site-specificity of arterial gene transfer, assessed by PCR, was not altered by administration of poloxamer 407. These findings suggest that poloxamer 407 may be useful to improve the efficiency of adenovirus-mediated arterial gene transfer.

Adenoviridae↗
PubMed1997

Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium.

In vitro studies suggest that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) may stimulate release of nitric oxide (NO) from endothelial cells. To investigate the hemodynamic consequences of recombinant VEGF/VPF administered in vivo, recombinant human VEGF/VPF was administered as a bolus dose of 500 micrograms to anesthetized (n = 6) or conscious (n = 5) New Zealand White rabbits, as well as anesthetized rabbits with diet-induced hypercholesterolemia (HC; n = 7). Anesthetized Yorkshire farm pigs (no specific dietary pretreatment) were studied before and after receiving 500 micrograms intravenous (IV; n = 5) or intracoronary (IC; n = 5) VEGF/VPF. In anesthetized, normal rabbits, mean arterial pressure (MAP) fell by 20.5 +/- 1.4% (P < .05 versus baseline) within 3 minutes after IV VEGF/VPF. Pretreatment with N omega-nitro-L-arginine caused a significant inhibition of VEGF/VPF-induced hypotension. In conscious, normal rabbits, VEGF/VPF produced a consistent though lesser reduction in MAP. The fall in MAP induced by VEGF/VPF in anesthetized, HC rabbits (21.5 +/- 2.5% from baseline) was no different from that observed in normal anesthetized rabbits. In pigs, both IV and IC administration of VEGF/VPF produced a prompt reduction in MAP. Heart rate increased, while cardiac output, stroke volume, left atrial pressure, and total peripheral resistance all declined to a similar, statistically significant degree in both IV and IC groups. Epicardial echocardiography disclosed neither global nor segmental wall motion abnormalities in response to VEGF/VPF. We conclude that (1) VEGF/VPF-stimulated release of NO, previously suggested in vitro, occurs in vivo; (2) this finding suggests that functional VEGF/VPF receptors are present on quiescent adult endothelium, consistent with a maintenance function for VEGF/VPF, which may include regulation of NO; and (3) the preserved response of HC rabbits suggests that endothelial cell receptors for VEGF/VPF are spared in the setting of hypercholesterolemia.

Animals↗
PubMed1997

Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty.

Intimal hyperplasia following angioplasty results in part from the migration and proliferation of vascular smooth muscle cells (VSMCs). However, the cell cycle regulatory networks underlying injury-induced VSMC proliferation are largely unknown. In the present study, we examined the kinetics of expression and activity of cell cycle regulatory factors after angioplasty in rat and human arteries. Cell lysates were prepared from uninjured rat carotid arteries and at different time points after balloon denudation. Marked induction of the proliferating cell nuclear antigen (PCNA), the G1/S cyclin-dependent kinase (cdk2), and its regulatory subunits (cyclin E and cyclin A) occurred between 1 and 2 days after angioplasty, was sustained up to 10 days after injury, and then declined. Induction of these factors correlated with increased cdk2-, cyclin E-, and cyclin A-dependent kinase activity, indicating the assembly of functional cdk2/cyclin E and cdk2/cyclin A holoenzymes in the injured arterial wall. Immunohistochemical analysis revealed early expression of cdk2, cyclin E, and PCNA within the media of injured carotid arteries. At later time points, expression of these markers declined to basal levels in the media but was detected within the intimal lesion. Thus, VSMC proliferation after angioplasty in the rat carotid artery is associated with a temporally and spatially coordinated expression of cdk2, cyclins E and A, and PCNA. Analysis of human arteries also revealed expression of these factors in VSMCs within restenotic lesions. Thus, cdk2 and its regulatory cyclins may be suitable targets to limit human restenosis.

Actins↗
PubMed1997

Arterial gene therapy: a molecular biological perspective for the treatment of arterial ischemia.

Since most cells do not naturally incorporate foreign DNA, strategies for arterial gene transfer have generally relied on agents designed to augment cellular DNA uptake such as liposomes, viral vectors, and protein conjugates. However, even when DNA has been successfully introduced into a cell, the likelihood of gene expression remains uncertain because the transgene has to escape lysosomal degradation, be transferred to the nucleus for transcription of mRNA, and be exported to the cytoplasm for translation and/or post-translation modification of the protein product. Transfection efficiency represents the final index of this regulatory sequence. The extent to which this compromises human gene therapy for cardiovascular disease depends in part on the presence or absence of a signal sequence permitting active secretion of the gene product. The implication of these issues is discussed with regard to restenosis and angiogenesis.

Animals↗