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J G Pickering

Publications and source records attributed to J G Pickering.

At least 37 records · Page 2Linked to original sources
PubMed1996

Intimal thickening develops without humoral immunity in a mouse aortic allograft model of chronic vascular rejection.

BACKGROUND: The major threat to the long-term survival of cardiac allograft recipients is the development of diffuse intimal thickening in the allograft coronary arteries through mechanisms that are poorly understood. Although antidonor antibodies have been associated with the development of this condition, a causal relationship has not been established. METHODS AND RESULTS: To determine whether humoral immune responses are necessary for the development of graft vascular disease, we performed abdominal aortic allografts from normal donor mice into different immunodeficient recipient mice: those lacking all donor-specific immune responses (severe combined immunodeficient [SCID] mice and recombination activating gene-1 [RAG-1]-deficient mice) and those lacking humoral immune responses alone owing to a targeted deletion of the joining region (JH) gene segments for the immunoglobulin heavy chain. At 6 to 9 weeks after transplantation, aortic allografts in normal immunocompetent recipients showed concentric intimal thickening extending the full length of the graft (percent luminal reduction, [%LR], 31.2 +/- 9.1 [mean +/- SD] and 38.5 +/- 3.6 in different donor-recipient strain combinations). In contrast, syngeneic (histocompatible) aortic grafts showed a normal-appearing vessel wall (%LR, 1.6 +/- 0.7). In both SCID and RAG-1-deficient recipients, aortic allografts showed a virtual absence of neointimal formation (%LR, 3.7 +/- 2.1 and 3.8 +/- 1.6 in SCID and RAG-1-deficient recipients, respectively), indicating a critical etiological role for alloimmune responses in this model. Importantly, allografts in JH-deficient mice showed marked intimal thickening (%LR, 35.7 +/- 7.9), with an appearance histologically indistinguishable from that of normal immunocompetent recipients. CONCLUSIONS: Neointimal formation in graft vascular disease is critically dependent on alloimmune responses of the host. Humoral effector mechanisms, however, may not be required.

Analysis of Variance↗
PubMed1996

Evidence for rapid accumulation and persistently disordered architecture of fibrillar collagen in human coronary restenosis lesions.

The pattern of collagen deposition after coronary angioplasty could significantly influence recurrent lesion formation. Traditional histologic assessments of coronary restenosis lesions have not identified abundant collagen fibers in restenotic tissue; however, these methods can suffer from lack of sensitivity and are not quantitative. We analyzed collagen architecture in 40 coronary lesions retrieved from patients by directional atherectomy, by exploiting the birefringent properties of fibrillar collagen. Picrosirius red-stained sections were illuminated with circularly polarized light, and fiber content and thickness were quantified by digital image analysis. Fifteen of 19 restenosis lesions (79%) and 1 of 21 native atherosclerosis lesions (5%) displayed a pattern of reactive intimal modeling, characterized by stellateshaped smooth muscle cells variably oriented in a loose extracellular matrix. There was an apparent paucity of collagen fibers in these regions based on staining with Movat's pentachrome, a traditional connective tissue stain. However, circular polarization light microscopy revealed an extensive distribution of collagen fibers in restenosis tissue, occupying 79.9% +/- 11.8% of the section area. Despite this high collagen content, the restenosis lesions were distinct from de novo atherosclerosis lesions in having a disordered collagen alignment, reduced fiber packing (p < 0.05), and thinner fibers (4.3 +/- 1.7 vs 9.2 +/- 4.3 microns, p < 0.001). Fiber diameter was greater in lesions retrieved between 3 and 17 months after angioplasty than in lesions retrieved between 1 week and 3 months (p < 0.05). However, fiber disorientation was evident in all lesions retrieved after 1 week, with little similarity to that of native plaque. Lesions retrieved within 1 week of angioplasty represented a distinct group with identical collagen features as in de novo atherosclerosis lesions, implying a different mechanism of restenosis in that population. We conclude that human coronary restenosis involves rapid accumulation of collagen fibers, which are persistently disordered. This may be critical in the development of restenosis and could significantly influence therapeutic attempts to control the process.

Atherectomy, Coronary↗
PubMed1996

Processing of chimeric antisense oligonucleotides by human vascular smooth muscle cells and human atherosclerotic plaque. Implications for antisense therapy of restenosis after angioplasty.

BACKGROUND: Antisense oligonucleotides have been used in animals to inhibit the accumulation of vascular smooth muscle cells (VSMCs) after arterial injury. This has raised prospects for an oligonucleotide-mediated approach to prevent restenosis in patients undergoing angioplasty. However, little is known about the processing of oligonucleotides by human VSMCs or their bioavailability in human atherosclerotic tissue. METHODS AND RESULTS: Oligonucleotides were synthesized with a mixture of unmodified and sulfur-modified linkages (S-chimeric oligonucleotides). These were more stable than unmodified oligonucleotides and could be recovered from within human VSMCs after 36 hours. Oligonucleotide antisense to human proliferating cell nuclear antigen mRNA specifically reduced DNA synthesis (P < .01) and proliferating cell nuclear antigen protein content (P < .05) in human VSMCs. Confocal microscopy of both live and fixed cells showed modest oligonucleotide uptake that was primarily nuclear. Surprisingly, cationic liposomes did not enhance nuclear uptake but led to extensive, punctated cytoplasmic loading without an enhanced antisense effect. Oligonucleotides incubated with human coronary atherosclerosis fragments associated with cells within 1 hour, despite the presence of abundant extracellular matrix. CONCLUSIONS: S-chimeric oligonucleotides are stable and can specifically inhibit gene expression in human VSMCs. Nuclear transport is a feature of oligonucleotide processing by human VSMCs, indicating a potential influence at the nuclear level rather than with cytoplasmic mRNA. Cationic liposomes increased oligonucleotide uptake but not intracellular bioavailability, and S-chimeric oligonucleotides can be incorporated into cells within human atherosclerotic plaque, despite the presence of a dense extracellular matrix.

Angioplasty↗
PubMed1996

Vascular applications of human gene therapy.

Complexing recombinant DNA with cationic liposomes is a convenient means of introducing foreign genes into cells (lipofection) and could potentially form the basis for genetically modifying diseased blood vessels in patients. The mechanism of lipofection is incompletely understood but it is recognized that the degree of successful gene transfer is highly dependent on cell type. We have transfected primary cultures of human vascular smooth muscle cells with a plasmid expressing either firefly luciferase (Luc) or nuclear-localized beta-galactosidase (NL-beta-gal). Cells were derived from either normal human internal mammary arteries, fragments of primary atherosclerotic plaque, or fragments of restenotic lesion. Concurrent lipofection of rabbit vascular smooth muscle cells and NIH 3T3 cells was performed as well. Compared to NIH 3T3 cells, expression in human vascular smooth muscle cells was markedly reduced: in cells derived from internal mammary artery, Luc expression, normalized for protein content, was 123-fold lower than in NIH 3T3 cells, while the proportion of cells expressing NL-beta-gal was 30-fold lower. Luc expression in cells derived from restenotic tissue was significantly greater than from cells derived from primary plaque. Within a given population of cells, the mitotic index of cells expressing the recombinant gene was significantly higher than the mitotic index for the total population of cells (p < 0.05). Finally, cotransfection experiments, in which lipofection of smooth muscle cells was performed using genes for NL-beta-gal and for human growth hormone, showed that among positive transfectants, a high proportion of cells (23-36%) co-expressed both genes. Thus, the efficiency of successful lipofection in human vascular smooth muscle cells in vitro is low. Transfection appears to be preferentially facilitated in cells derived from restenotic tissue and specific properties of smooth muscle cells, including growth rates, appear to be critical for successful transfection. Further elucidation of cell properties that promote transfection is required to augment the efficiency of liposome-mediated gene transfer in human vascular cells.

Adenoviridae↗
PubMed1995

Epidermal growth factor receptor-targeted cytotoxin inhibits neointimal hyperplasia in vivo. Results of local versus systemic administration.

Smooth muscle cell accumulation is a key feature of restenosis that may be inhibited by the delivery of receptor-targeted cytotoxins. DAB389EGF is a recombinant fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor (EGF). We investigated the effectiveness of DAB389EGF to inhibit neointimal hyperplasia in the balloon-injured rat carotid artery. Incubation of rat carotid arteries with 125I-labeled EGF revealed extensive EGF binding sites in the neointima of balloon-injured arteries. Sixty rats subsequently received either saline or DAB389EGF (total dose, 0.15 mg) delivered immediately following balloon injury either systemically, via 14-day continuous osmotic pump infusion, or locally, via 30-minute intraluminal incubation. The effect of both treatment strategies was measured 2 weeks after injury by cross-sectional morphometric analysis of intimal area, the ratio of intimal/medial area (I/M), and the percent luminal narrowing (%LN). In addition, proliferative activity was assessed by immunostaining for the presence of the proliferating cell nuclear antigen (PCNA). Compared with controls, systemic delivery of fusion toxin significantly reduced intimal area, I/M, and %LN by 40%, 40%, and 29%, respectively. However, these rats exhibited 2% weight loss, indicating mild systemic toxicity. Local, intraluminal administration of DAB389EGF yielded a more pronounced reduction in intimal area, I/M, and %LN by 74%, 79%, and 72%, respectively. This inhibitory effect was preserved at 3 weeks postinjury, and PCNA immunostaining of locally treated arteries revealed a virtual absence of proliferative activity in the neointima and media at this timepoint. In contrast to systemically treated rats, rats receiving fusion toxin locally gained weight at a rate similar to controls, indicating avoidance of systemic toxicity. We conclude that DAB389EGF is a potent inhibitor of neointimal hyperplasia in vivo and that whereas an inhibitory effect may be achieved by systemic delivery, local delivery appears to be more potent, avoids systemic toxicity, and thus represents a feasible strategy to preempt restenosis.

Animals↗
PubMed1994

Oxygen uptake kinetics in cardiac transplant recipients.

Our purpose was to examine the gas exchange response to exercise in heart transplant (HT) patients and to characterize the O2 uptake kinetics (tau VO2) during successive square-wave on-transients from loadless cycling to moderate exercise. We hypothesized that with a slow heart rate response (and O2 transport limitation) O2 kinetics would be slowed but that with a repeated exercise initiated while the heart rate remained elevated the tau VO2 would be faster. Six male HT patients performed two ramp-function tests to determine peak O2 uptake (1.32 +/- 0.23 l/min) and ventilation threshold (1.02 +/- 0.16 l/min). Patients subsequently completed two repeats of a square-wave forcing function and repeated this on 2 days. Alveolar gas exchange was measured breath by breath. A monoexponential fit of signal-averaged data of the first exercise on-transient (between days) yielded a significantly slower tau VO2 in HT subjects than in healthy men (mean age 47 yr; n = 8) (77 +/- 26 vs. 45 +/- 4 s). With successive exercise (2nd transition) initiated while HR remained elevated the tau VO2 of HT patients was 46 +/- 17 s. The faster O2 kinetics of the second transition suggests that O2 delivery was enhanced and therefore that the tau VO2 may reflect bioenergetic processes controlling the rate of oxidative metabolism.

Blood Gas Analysis↗
PubMed1994

Liposome-mediated gene transfer into human vascular smooth muscle cells.

BACKGROUND: Complexing recombinant DNA with cationic liposomes is a convenient means of introducing foreign genes into cells (lipofection) and could potentially form the basis for genetically modifying diseased blood vessels in patients. The mechanism of lipofection is incompletely understood, but it is recognized that the degree of successful gene transfer is highly dependent on cell type. To date, there has been no reported experience with lipofection of human vascular smooth muscle cells. METHODS AND RESULTS: Primary cultures of human vascular smooth muscle cells were transfected under optimized conditions with a plasmid expressing either firefly luciferase (Luc) or nuclear-localized beta-galactosidase (NL-beta-gal). Cells were derived from either normal human internal mammary arteries (n = 6), fragments of primary atherosclerotic plaque (n = 4), or fragments of restenotic lesions (n = 5). Concurrent lipofection of rabbit vascular smooth muscle cells and NIH 3T3 cells was performed as well. Cultures derived from 15 patients all demonstrated positive expression of the reporter gene. Compared with NIH 3T3 cells, however, expression in human vascular smooth muscle cells was markedly reduced: in cells derived from internal mammary artery, Luc expression, normalized for protein content, was 123-fold lower than in NIH 3T3 cells, whereas the proportion of cells expressing NL-beta-gal was 30-fold lower. Luc expression in cells derived from restenotic tissue was significantly greater than from cells derived from primary plaque (P < .03). Within a given population of cells, the mitotic index of cells expressing the recombinant gene was significantly higher than the mitotic index for the total population of cells (P < .05). Finally, cotransfection experiments, in which lipofection of smooth muscle cells was performed using genes for NL-beta-gal and for human growth hormone, showed that among positive transfects, a high proportion of cells (23% to 36%) coexpressed both genes. CONCLUSIONS: The efficiency of successful lipofection in human vascular smooth muscle cells in vitro is low. Transfection appears to be preferentially facilitated in cells derived from restenotic tissue, and specific properties of smooth muscle cells, including growth rates, appear to be critical for successful transfection. Further elucidation of cell properties that promote transfection is required to augment the efficiency of liposome-mediated gene transfer in human vascular cells.

3T3 Cells↗
PubMed1994

Use of the rabbit ear artery to serially assess foreign protein secretion after site-specific arterial gene transfer in vivo. Evidence that anatomic identification of successful gene transfer may underestimate the potential magnitude of transgene expression.

BACKGROUND: The development of molecular strategies for the treatment of restenosis has been hindered by low efficiencies of in vivo arterial transfection. Expression of intracellular marker proteins is generally evident in < 1% of vascular smooth muscle cells after in vivo arterial transfection. Efforts to improve the efficiency of in vivo gene transfer have been further impeded by the use of transgenes encoding for intracellular marker proteins, necessitating tissue removal and limiting survey for expression to one point in time. METHODS AND RESULTS: To study gene expression on a serial basis in vivo and determine the relation between a secreted gene product and transfection efficiency after in vivo arterial gene transfer, a method for performing and serially monitoring gene expression in vivo was developed using the central artery of the rabbit ear. Liposome-mediated transfection of plasmid DNA containing the gene for human growth hormone (hGH) was successfully performed in 18 of 23 arteries. Serum hGH levels measured 5 days after transfection ranged from 0.1 to 3.8 ng/mL (mean, 0.97 ng/mL); in contrast, serum drawn from the control arteries demonstrated no evidence of hGH production. Serial measurement of hGH from transfected arteries demonstrated maximum hGH secretion 5 days after transfection and no detectable hormone after 20 days. Despite these levels of secreted gene product documented in vivo, immunohistochemical staining of sections taken from the rabbit ear artery at necropsy disclosed only rare cells in which there was evidence of successful transfection. CONCLUSIONS: These experiments demonstrate a useful method of performing serial in vivo analyses of gene expression after vascular transfection and that anatomic analyses of transfection efficiency may underestimate the potential magnitude of expression in the case of a secreted gene product. These findings have implications for the clinical application of somatic gene therapy because low-efficiency transfection with a gene encoding for a secreted protein may achieve therapeutic effects not realized by transfection with genes encoding for proteins that remain intracellular.

Animals↗
PubMed1994

Increased gene expression after liposome-mediated arterial gene transfer associated with intimal smooth muscle cell proliferation. In vitro and in vivo findings in a rabbit model of vascular injury.

Arterial gene transfer represents a novel strategy that is potentially applicable to a variety of cardiovascular disorders. Attempts to perform arterial gene transfer using nonviral vectors have been compromised by a low transfection efficiency. We investigated the hypothesis that cellular proliferation induced by arterial injury could augment gene expression after liposome-mediated gene transfer. Nondenuded and denuded rabbit arterial strips were maintained in culture for up to 21 d, after which transfection was performed with a mixture of the plasmid encoding firefly luciferase and cationic liposomes. In non-denuded arteries, the culture interval before transfection did not affect the gene expression. In contrast, denuded arteries cultured for 3-14 d before transfection yielded 7-13-fold higher expression (vs. day 0; P < 0.005). Transfection was then performed percutaneously to the iliac arteries of live rabbits with or without antecedent angioplasty. Gene expression increased when transfection was performed 3-7 d postangioplasty (P < 0.05). Proliferative activity of neointimal cells assessed in vitro by [3H]thymidine incorporation, and in vivo by immunostaining for proliferating cell nuclear antigen, increased and declined in parallel with gene expression. These findings thus indicate that the expression of liposome-mediated arterial gene transfer may be augmented in presence of ongoing cellular proliferation.

Animals↗
PubMed1993

Excimer laser-induced vasoreactivity.

Earlier generations of excimer lasers, designed for industrial or non-cardiovascular medical applications, have been previously shown consistently to induce vasorelaxation of vascular smooth muscle in vitro. Such lasers were typically characterized by pulse durations of < or = 15 nanoseconds (ns). Excimer lasers currently employed for cardiovascular applications were designed with longer pulse durations (up to 220 ns) to facilitate fibreoptic transmission. Because arterial spasm has been observed in patients undergoing percutaneous revascularization with such lasers, we investigated the effect of so-called 'stretched pulse' excimer laser irradiation on vasomotor reactivity. A total of 69 rings of aorta harvested from New Zealand white rabbits were mounted isometrically in Krebs buffer solution and exposed to 308 nm from an excimer laser with a pulse duration of 120 ns. Fifty rings were exposed without pharmacological pre-treatment. The remaining 19 rings were exposed after pharmacological pre-treatment: 11 were precontracted with norepinephrine (NE, 10(-9)-10(-5) M), while eight were irradiated in Ca(2+)-free buffer after pre-relaxation with nitroglycerin (NTG, 7 x 10(-5) M). Without pharmacological pre-treatment, the vasomotor response to the excimer laser was variable: vasoconstriction was observed in 27 rings (16.1 +/- 0.8% (mean +/- SEM) of response to 5-HT), vasorelaxation in 21 rings (43.2 +/- 17.0% of response to 5-HT), and a heterogeneous response (vasoconstriction 4.9 +/- 1.0%, vasorelaxation 12.9 +/- 0.3%) in two rings. The vector of vasomotor response in non-precontracted rings was not predicted by fluence, frequency or temperature rise. A consistent vasomotor response was recorded only when pharmacological pre-treatment was employed. Among 11 rings pre-contracted with NE, the excimer laser produced vasorelaxation in 34/34 (100%) exposures; in contrast, among eight rings pre-relaxed with NTG in Ca(2+)-free buffer, the excimer laser produced vasoconstriction in 40/40 (100%) exposures. For all rings, including pre-contracted, pre-relaxed and those which were not pharmacologically pre-treated, the vector of vasomotor response was endothelium-independent. The magnitude of all vasomotor responses, including vasoconstriction in non-precontracted rings, could be diminished by limiting the duration of exposure. Thus, in contrast to the earlier generation, short-pulse excimer lasers, long pulse-duration excimer lasers in current clinical trials produce an unpredictable, heterogeneous vasomotor response. This in-vitro finding is consistent with the unpredictable development of vascular spasm in patients undergoing excimer laser angioplasty. Furthermore, these findings support the concept of employing abridged pulse trains to diminish the likelihood of laser-induced vasoconstriction during excimer laser angioplasty.(ABSTRACT TRUNCATED AT 400 WORDS)

Angioplasty, Laser↗
PubMed1993

Prevention of smooth muscle cell outgrowth from human atherosclerotic plaque by a recombinant cytotoxin specific for the epidermal growth factor receptor.

Smooth muscle cell proliferation in the intima of arteries is a principal event associated with vascular narrowing after balloon angioplasty and bypass surgery. Techniques for limiting smooth muscle cell proliferation, however, have not as yet yielded any therapeutic benefit for these conditions. This may reflect the present lack of sufficiently potent and specific inhibitors of smooth muscle cell proliferation. DAB389 EGF is a genetically engineered fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor. We evaluated the effect of this fusion toxin on human vascular smooth muscle cells in culture. Incubation of proliferating cells with DAB389 EGF yielded a dose-dependent inhibition of protein synthesis, as assessed by uptake of [3H]leucine, with an IC50 of 40 pM. The cytotoxic effect was inhibited in the presence of excess EGF or with monoclonal antibody to the EGF receptor. We further studied the effect of the fusion toxin on smooth muscle cell outgrowth from human atherosclerotic plaque. Outgrowth was markedly inhibited after as little as 1 h of exposure to the fusion protein. Furthermore, complete inhibition of proliferation of cells within the plaque could be attained. These results demonstrate that DAB389 EGF is highly cytotoxic to human smooth muscle cells proliferating in culture and can prevent smooth muscle cell outgrowth from "growth-stimulated" human atherosclerotic plaque. DAB389 EGF may therefore be of therapeutic value in vascular diseases characterized by smooth muscle cell accumulation.

Arteriosclerosis↗
PubMed1993

Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization.

We evaluated the proliferative activity of human atherosclerotic lesions associated with active symptoms of ischemia, by assessing the expression of the proliferating cell nuclear antigen (PCNA). We confirmed in vitro that PCNA, an essential component of the DNA synthesis machinery, is selectively expressed in proliferating human vascular smooth muscle cells. 37 atherosclerotic lesions (18 primary and 19 restenotic) retrieved by directional atherectomy from either coronary or peripheral arteries were then studied for the expression of PCNA, using in situ hybridization or immunohistochemistry. Among plaques studied by in situ hybridization, 7 out of 11 primary and 11 out of 11 restenotic lesions contained PCNA-positive cells. The mean rate of proliferation (percent of PCNA-positive cells) was 7.2 +/- 10.8% in primary lesions and 20.6 +/- 18.2% in restenotic lesions (P < 0.05). Among specimens studied by immunohistochemistry, five out of seven primary and eight out of eight restenotic lesions contained proliferating cells. The mean rate of proliferation was again higher in the restenotic (15.2 +/- 13.6%) than primary (3.6 +/- 3.5%) lesions (P < 0.05). Proliferating cells were detected as late as 1 yr after angioplasty. We conclude that cellular proliferation is a feature of atherosclerotic lesions which are associated with symptoms of ischemia, but that it is more prominent in restenosis compared to primary lesions. These findings have implications for therapies aimed at limiting lesion growth, particularly after percutaneous revascularization.

Adult↗
PubMed1993

Direct myocardial transfection in two animal models. Evaluation of parameters affecting gene expression and percutaneous gene delivery.

BACKGROUND: Gene therapy represents a novel approach to the treatment of a variety of disease states. Direct injection of pure untreated DNA into skeletal and cardiac muscle is sufficient to perform gene transfer in vivo. Little information is available, however, regarding the extent to which individual parameters of direct gene delivery affect the efficiency of myocardial transfection. Moreover, the fact that all previously reported studies of successful myocardial transfection were performed using open chest surgery to visualize the heart for direct injection of DNA constitutes a potential limitation to clinical applications of myocardial gene transfer. The objectives of the present study were: (a) to determine the extent to which gene expression is altered by varying the amount of DNA and the volume of the delivery vehicle; (b) to study whether protracted expression of DNA persists after direct myocardial transfection; and (c) to test the feasibility of percutaneous, transthoracic myocardial injection of DNA. EXPERIMENTAL DESIGN: New Zealand white rabbits (N = 30) were transfected with the firefly (Photinus pyralis) luciferase reporter gene. Twenty-three rabbits were segregated into 5 groups according to the amount of DNA and volume of delivery vehicle injected. These rabbits were sacrificed at 5 days posttransfection to measure the level of luciferase gene expression. Three more rabbits were designated exclusively for in situ hybridization. Two rabbits were sacrificed at 5 and 6 months, respectively to evaluate long-term expression. Two additional rabbits were used as controls. Yucatan microswine (N = 4) were used to evaluate the feasibility of percutaneous, transthoracic myocardial transfection with beta-galactosidase or luciferase reporter genes. Microswine were sacrificed immediately post-sham transfection (N = 2), or 5 days postmyocardial transfection (N = 2). RESULTS: Among 11 rabbits, transfection with 10 micrograms of DNA (N = 4), 25 micrograms of DNA (N = 3), and 50 micrograms of DNA (N = 4) in 100 microL of injectate yielded a step-wise but statistically insignificant increase in luciferase activity. Among 16 rabbits, transfection with 50 micrograms of DNA in injectate volumes of 50 microliters (N = 4), 100 microliters (N = 4), 150 microliters (N = 4), and 300 microliters (N = 4) yielded a statistically significant (p < 0.05) increase in luciferase activity in those hearts transfected with 300 microliters compared with those hearts transfected with either 50 or 100 microliters. Luciferase activity at 5 and 6 months postmyocardial transfection was 7.14 and 68.8 Turner light units, respectively. In situ hybridization confirmed that myocytes represented the site of luciferase expression. Percutaneous myocardial transfection was successfully accomplished with both reporter genes. CONCLUSIONS: The results of the present study demonstrate that increasing the amount of DNA employed for direct myocardial transfection fails to produce a statistically significant increase in the level of gene expression. In contrast, increasing the volume of injectate used to directly transfect a constant amount of DNA (50 micrograms) produced significantly augmented expression of the reporter luciferase gene. Luciferase expression was detected 6 months posttransfection. Successful transfection after fluoroscopic-guided direct percutaneous delivery suggests that it may be feasible to deliver genetic material into the myocardium of patients in a similar fashion.

Animals↗
PubMed1992

Smooth muscle cell outgrowth from human atherosclerotic plaque: implications for the assessment of lesion biology.

OBJECTIVES: The purpose of this study was to determine whether the kinetics of smooth muscle cell outgrowth from in vitro explants of human atherosclerotic tissue is dependent on the nature of the atherosclerotic lesion in vivo. BACKGROUND: The use of techniques for percutaneous in vivo extraction of atherosclerotic plaque has provided the opportunity to study human atheroma-derived smooth muscle cells in culture. However, because of cell selection and changes in phenotype, in vitro findings may not always reflect the biologic properties of the vessel wall, particularly if cells are in culture for prolonged periods. In contrast, studies with nonhuman cells have suggested that the rate at which cells grow out of tissue explants is closely related to the status of the cells in vivo. METHODS: Atherosclerotic tissue from 41 lesions, including primary plaques (from peripheral arteries and venous bypass conduits) and restenotic lesions (from peripheral arteries and venous conduits) were divided into a total of 1,596 fragments and placed in culture on fibronectin-coated wells. Explant outgrowth was scored over the ensuing 1 month to determine the prevalence and time course of smooth muscle cell outgrowth and the total cellular accumulation. RESULTS: Explant fragments from 40 (98%) of the 41 lesions yielded an outgrowth of smooth muscle cells, confirmed by immunocytochemistry. The mean proportion of adherent explant fragments yielding outgrowth, per lesion, was 69 +/- 4% and was higher in restenotic tissue (81 +/- 3%) than in primary tissue (56 +/- 6%, p < 0.001). For primary lesions, initiation of outgrowth was half-maximal by 8.7 +/- 0.4 days; for restenotic explants, initiation of outgrowth was considerably faster (half-maximal by 5.9 +/- 0.6 days, p < 0.001). Similarly, accumulation of smooth muscle cells around an explant was significantly greater for restenotic lesions (2,791 +/- 631 cells/explant) than for primary lesions (653 +/- 144 cells/explant, p < 0.01). Labeling of first-passage cells with [3H]-thymidine indicated that cells from restenotic lesions had a 1.3-fold greater incorporation rate than did cells from primary lesions (p < 0.05). CONCLUSIONS: Smooth muscle cells may be reliably cultivated by explant outgrowth from a variety of human atherosclerotic plaque types obtained intraoperatively or percutaneously. The kinetics of outgrowth from restenotic tissue is distinctly different from that of outgrowth from primary tissue, suggesting a relation between the in vitro outgrowth behavior and the biology of the lesion in vivo. Assessment of smooth muscle cell outgrowth from human atherosclerotic plaque may thus represent a practical and reliable means to investigate the biologic behavior, including growth characteristics, of individual atherosclerotic lesions from human subjects. This technique may also offer a suitable assay system for evaluating therapies designed to inhibit lesion proliferation.

Analysis of Variance↗
PubMed1992

Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions.

Human atheromata obtained in vivo were used to test the hypothesis that transforming growth factor-beta 1 plays a role in the development of vascular restenosis. We analyzed 28 specimens from patients with primary atherosclerotic or restenotic lesions; 26 of these were obtained by directional atherectomy and 2 at the time of coronary bypass surgery. Seven control tissues included operatively excised segments of human internal mammary artery, myocardium, and unused portions of vein graft obtained intraoperatively. From these 35 specimens, 210 sections were examined using in situ hybridization. Measurement of silver grains/nucleus disclosed that expression of transforming growth factor-beta 1 mRNA was highest in restenotic tissues (P < 0.001 vs. primary atherosclerotic tissues) and lowest in nonatherosclerotic (control) tissues. In cultures of human vascular smooth muscle cells grown from explants of internal mammary artery, expression of mRNA for transforming growth factor-beta 1 was significantly greater in subconfluent than in confluent smooth muscle cells (P = 0.05). Transforming growth factor type-beta III receptor was expressed in cell cultures and undetectable in the tissue specimens. Sections taken adjacent to those studied by in situ hybridization were examined by immunohistochemistry using antibodies against transforming growth factor-beta 1 and alpha-actin (as a marker for smooth muscle cells) and disclosed transforming growth factor-beta 1 in smooth muscle cells present in these sections. These findings are consistent with the concept that transforming growth factor-beta 1 plays an important role in modulating repair of vascular injury, including restenosis, after balloon angioplasty.

Actins↗
PubMed1991

Coronary capillaries in patients with congestive cardiomyopathy or angina pectoris with patent main coronary arteries. Ultrastructural morphometry of endomyocardial biopsy samples.

BACKGROUND: The coronary microvasculature may be abnormal even in the presence of angiographically normal epicardial arteries. Abnormalities of small coronary vessels have been invoked as a cause of angina. METHODS AND RESULTS: To quantitatively evaluate the morphology of capillaries in patients with idiopathic dilated cardiomyopathy (DCM) or the syndrome of angina and small vessel disease (SVD), we performed electron microscopic morphometry of capillaries in right ventricular biopsy samples taken from 32 patients. Ten had angina, normal epicardial coronary arteries, and evidence for SVD; 12 had DCM; and 10 had normal hearts. In patients with DCM, the ratio of microvessels to myocytes was not different than that of controls (0.49 +/- 0.06 versus 0.51 +/- 0.05). Mean cross-sectional areas of the capillaries (lumen plus wall) and lumen were significantly greater than those of controls (45.3 +/- 15.1 versus 22.7 +/- 8.3 micron 2, p less than 0.001; 17.6 +/- 6.9 versus 11.6 +/- 6.2 micron 2, p less than 0.05, respectively). Fibrous content of the myocardium, as assessed by quantitative light microscopy, was significantly increased (16.3 +/- 3.3% versus 5.0 +/- 2.4%, p less than 0.001). In contrast, in patients with SVD, the capillary-to-myocyte ratio was reduced (0.33 +/- 0.08, p less than 0.001). Although mean cross-sectional areas of the entire capillary (32.4 +/- 19.7 micron 2) and the lumen (8.9 +/- 7.8 micron 2) were not statistically different than those of controls, there was an absence of capillaries less than 15 micron 2 in cross-sectional area, and the frequency distribution of the lumen area was skewed to the left. Swollen endothelial cells frequently encroached upon the lumen. There was a mild increase in fibrous content (9.5 +/- 3.7%, p less than 0.05). CONCLUSIONS: Enlarged capillaries and a normal ratio of capillaries to myocytes appear to be features of DCM. Of the patients with SVD, there was both a relative lack of capillaries and capillary lumen narrowing from swollen endothelium. These changes may induce ischemia and angina and may result in mild fibrosis.

Adult↗