Lawrence Weir

Arterial Gene Therapy for Therapeutic Angiogenesis in Patients With Peripheral Artery Disease

### Peripheral Artery Disease: Primary Pharmacological Therapy Is Ineffective for Patients With Critical Limb Ischemia The prognosis for patients with chronic critical leg ischemia, ie, rest pain and/or established lesions that jeopardize the integrity of the lower limbs, is often poor. Psychological testing of such patients has typically disclosed quality-of-life indexes similar to those of patients with cancer in critical or even terminal phases of their illness.1 It has been estimated that in toto,2 150 000 patients per year require lower-limb amputations for ischemic disease in the United States. Their prognosis after amputation is even worse3 : the perioperative mortality for below-knee amputation in most series is 5% to 10% and for above-knee amputation 15% to 20%. Even when they survive, nearly 40% will have died within 2 years of their first major amputation; a major amputation is required in 30% of cases; and full mobility is achieved in only 50% of below-knee and 25% of above-knee amputees. These grim statistics are compounded by the lack of efficacious drug therapy. As concluded in the Consensus Document of the European Working Group on Critical Leg Ischemia,3 “. . .there presently is inadequate evidence from published studies to support the routine use of primary pharmacological treatment in patients with critical leg ischemia. . . .” Evidence for the utility of medical therapy in the treatment of claudication is no better.4 5 Consequently, the need for alternative treatment strategies in such patients is compelling. ### Therapeutic Angiogenesis Is a Novel Strategy for the Treatment of Critical Limb Ischemia The therapeutic implications of angiogenic growth factors were identified by the pioneering work of Folkman6 and other workers more than two decades ago. Beginning a little more than a decade ago,7 a series of polypeptide growth factors were purified, sequenced, and demonstrated to be responsible for natural as well as pathological angiogenesis. More recent investigations have established the feasibility of using recombinant formulations of such angiogenic growth …

Relation between Activated Smooth-Muscle Cells in Coronary-Artery Lesions and Restenosis after Atherectomy

BACKGROUND Neointimal proliferation leading to restenosis frequently develops after coronary angioplasty. This process is associated with a change in vascular smooth-muscle cells from a contractile (quiescent) phenotype to a synthetic or proliferating (activated) one. We investigated whether the presence of activated smooth-muscle cells in coronary lesions at the time of coronary atherectomy predisposes patients to subsequent restenosis. METHODS We used in situ hybridization to study the expression of messenger RNA in coronary-atherectomy specimens from 20 patients. Plaque material was hybridized with a probe for the B isoform of human nonmuscle myosin heavy chain, a major nonmuscle myosin isoform in activated, but not quiescent, smooth-muscle cells. Angiographic follow-up data were obtained a mean (+/- SD) of 174 +/- 54 days after atherectomy in 16 of the 20 patients, and the extent of recurrent luminal narrowing was analyzed quantitatively. The presence of restenosis was assessed by exercise thallium scintigraphy in the other four patients. RESULTS Atherectomy specimens from 10 of the 20 patients showed hybridization with the probe, defined as the clustering of more than 20 silver grains per cell nucleus in more than 10 nuclei in five high-power fields (x250); specimens from the other 10 patients showed no such hybridization. At follow-up, restenosis had developed in 8 of the 10 patients with positive hybridization results, but was absent in 9 of the 10 patients with negative results (P = 0.007). The degree of late loss in luminal diameter was significantly higher in patients with positive hybridization results than in those with negative results (ratio of late loss to immediate gain after atherectomy, 0.76 +/- 0.3 vs. 0.36 +/- 0.3; P < 0.001). CONCLUSIONS We conclude that the expression of the B isoform of nonmuscle myosin heavy chain is increased in some coronary atherosclerotic plaques and that this increase in expression identifies a group of lesions at high risk for restenosis after atherectomy.

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.

BackgroundThe 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 ResultsTo 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 proene duction. 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. ConclusionsThese 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.

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.

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.

Evidence implicating nonmuscle myosin in restenosis. Use of in situ hybridization to analyze human vascular lesions obtained by directional atherectomy.

BackgroundIdentification of genes that are specifically activated in restenosis lesions after percutaneous transluminal angioplasty represents a necessary step toward molecular manipulation designed to inhibit cellular proliferation responsible for such lesions. Whereas quiescent smooth muscle cells (contractile phenotype) preferentially express smooth muscle myosin, proliferating smooth muscle cells (synthetic phenotype) have been shown to preferentially express nonmuscle myosin in vitro. Accordingly, we analyzed the expression of a recently cloned isoform of human nonmuscle myosin heavy chain (MHC-B) in fresh human restenotic lesions. Methods and ResultsA total of 10 lesions, including four restenosis (three superficial femoral arterial lesions and one saphenous vein bypass lesion) and six primary (four superficial femoral arterial lesions and two coronary arterial lesions) obtained percutaneously by directional atherectomy, were processed for examination by in situ hybridization. In total, 150 tissue sections of restenotic lesions (66 sections), primary lesions (78 sections), and normal internal mammary artery (six sections) were hybridized with the nonmuscle MHC-B probe. Restenotic lesions showed intense hybridization to the nonmuscle MHC-B cRNA probe, as demonstrated by a clustering of more than 20 grains per cell nucleus in 80% of the cells examined within a high-power field (×250); in contrast, an equivalent degree of hybridization was observed in only 7% of cells within primary lesions (p<0.001). Results of immunocytochemistry using monoclonal antibody to smooth muscle actin indicated that cells demonstrating strong hybridization were smooth muscle in origin. ConclusionsThese findings demonstrate that 1) human vascular tissue obtained by percutaneous directional atherectomy constitutes appropriate biopsy material for gene expression studies at the mRNA level, and 2) nonmuscle MHC-B mRNA is present in greater abundance among restenotic versus primary vascular stenoses. These observations thus provide a rational basis to explore restenotic lesions on a larger scale to identify genes that are activated in these lesions and establish potential targets for future gene therapy.

Use of human tissue specimens obtained by directional atherectomy to study restenosis.

Directional atherectomy has provided the opportunity to study the pathology of restenosis in human tissue specimens from live patients. The restenosis lesion is characterized by two distinctive features: a focus of hypercellularity, comprised of cells with phenotypic features of proliferative vascular smooth muscle cells (SMCs), and a rich, loose extracellular matrix (ECM). Analysis of restenosis lesions by in situ hybridization, immunohistochemistry, and cell culture has disclosed evidence of activated SMCs, and in many cases-particularly lesions from the peripheral vasculature-ongoing cellular proliferation. The ECM of restenosis lesions is biglycan rich and decorin poor, a finding that is associated with increased expression of transforming growth factor beta (TGF-β). While certain restenosis lesions contain foci of microangiogenesis, the pathogenetic significance of this feature remains uncertain.

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.

Persistently increased expression of the transforming growth factor-β1 gene in human vascular restenosis: Analysis of 62 patients with one or more episode of restenosis☆

Transforming growth factor-beta-1 (TGF-β1) is a multifunctional cytokine with both growth-promoting and growth-inhibiting properties. Moreover, there is abundant evidence that TGF-β1 is the principal growth factor responsible for regulating proteoglycan synthesis in human blood vessels. To determine the potential contribution of TGF-β1 to restenosis, the current investigation sought to determine the time course of expression postangioplasty of the TGF-β1 gene. In situ hybridization was performed on tissue specimens obtained by directional atherectomy from 62 patients who had previously undergone angioplasty of native coronary or peripheral arteries and/or saphenous vein bypass grafts. The time interval between angioplasty and atherectomy was 1 hour to 25 months (M ± SEM = 5 ± 4 months) for all 62 patients, 5 ± 4 months for coronary arterial specimens, 8 ± 5 months for vein graft specimens, and 7 ± 3 months for peripheral arterial specimens. TGF-β1 mRNA expression remained persistently increased independent of the site from or time interval following which the specimen was obtained. For saphenous vein by pass grafts, TGF-β1 expression was highest in specimens retreived from patients with multiple versus single episodes of restenosis (16 ± 5 vs. 6 ± 5 grains/nucleus, p < 0.01). TGF-β1 expression did not correlate with patient age, sex, or known risk factors for coronary heart disease. The persistently augmented expression of TGF-β1 observed in the present series of restenosis lesions provides further support for the concept that TGF-β1 influences growth and development of restenosis plaque.

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.