The axis of interleukin 12 and gamma interferon regulates acute vascular xenogeneic rejection.

Recent advances using transgenic animals or exogenous complement inhibitors have demonstrated prevention of hyperacute rejection of vascularized organs, but not graft loss due to acute vascular rejection. Using various wild-type and cytokine-deficient mice strains, we have examined the mechanisms of acute vascular rejection. C57BL/6 mice deficient in interleukin12 or gamma interferon showed faster acute vascular rejection than did wild-type mice. Furthermore, mice defective in B-cell development showed no acute vascular rejection. These results demonstrate that the axis of interleukin 12 and gamma interferon provides a survival advantage in vascularized xenografts by delaying or preventing acute vascular rejection caused by a B cell-dependent mechanism.

Evidence for a role of collagen synthesis in arterial smooth muscle cell migration.

Migration of smooth muscle cells (SMCs) and collagen synthesis by SMCs are central to the pathophysiology of vascular disease. Both processes can be induced shortly after vascular injury; however, a functional relationship between them has not been established. In this study, we determined if collagen synthesis was required for SMC migration, using ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl-4-hydroxylase, and 3,4-DL-dehydroproline (DHP), a proline analogue, which we demonstrate inhibit collagen elaboration by porcine arterial SMCs. SMCs exposed to EDHB or DHP attached normally to collagen- and vitronectin-coated substrates; however, spreading on collagen but not vitronectin was inhibited. SMC migration speed, quantified by digital time-lapse video microscopy, was significantly and reversibly reduced by EDHB and DHP. Flow cytometry revealed that expression of beta1 integrins, through which SMCs interact with collagen, was unaffected by EDHB or DHP. However, both inhibitors prevented normal clustering of beta1 integrins on the surface of SMCs, consistent with a lack of appropriate matrix ligands for integrin engagement. Moreover, there was impaired recruitment of vinculin into focal adhesion complexes of spreading SMCs and disassembly of the smooth muscle alpha-actin-containing cytoskeleton. These findings suggest that de novo collagen synthesis plays a role in SMC migration and implicates a mechanism whereby newly synthesized collagen may be necessary to maintain the transcellular traction system required for effective locomotion.

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.

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.

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.

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.

Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery.

Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105+/-9 micrometer in length, and had a doubling time of 39+/-2 hours. Spindle-shaped clones (n=3) were larger (267+/-18 micrometer long, P<0.01) and grew slower (doubling time 65+/-4 hours, P<0.01). Both types of clones expressed smooth muscle (SM) alpha-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca(2+) transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM alpha-actin-positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.

Heat shock protein 47 is expressed in fibrous regions of human atheroma and Is regulated by growth factors and oxidized low-density lipoprotein.

BACKGROUND: Heat shock protein 47 (Hsp47) is a stress protein that may act as a chaperone for procollagen. Its involvement in atherosclerosis is unknown. METHODS AND RESULTS: Hsp47 expression in human coronary arteries was assessed by immunostaining. Strong focal expression was evident in atherosclerotic, but not normal, arteries and was prevalent in the collagenous regions. Double immunostaining revealed that all cells expressing type I procollagen also expressed Hsp47. Moreover, parallel regulation of proalpha1(I)collagen and Hsp47 mRNA expression occurred with cultured human smooth muscle cells stimulated with transforming growth factor-beta1 or fibroblast growth factor-2. However, a proportion of Hsp47-expressing cells in plaque did not express type I procollagen, and this pattern could be reproduced in culture. Heat shock and oxidized LDL stimulated the expression of Hsp47 mRNA by smooth muscle cells, without a concomitant rise in proalpha1(I)collagen expression. CONCLUSIONS: These findings identify Hsp47 as a novel constituent of human coronary atheroma. Its localization to the fibrous cap, regulation by growth factors in parallel with type I procollagen, and selective upregulation by stress raise the possibility that Hsp47 is a determinant of plaque stability.

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.

HSP47 expression by smooth muscle cells is increased during arterial development and lesion formation and is inhibited by fibrillar collagen.

HSP47 is a heat-shock protein that interacts with intracellular procollagen. It has been found in fibrous atherosclerotic plaque, but its involvement in acute vascular restructuring is unknown. We analyzed the expression of HSP47 and its regulation in the developing rat aorta and after balloon injury to the adult rat carotid artery. HSP47 was strongly expressed in each layer of the maturing fetal aorta (embryonic day 17 to birth). Expression declined during the first 4 postnatal days but persisted at low abundance into adulthood. HSP47 expression was substantially upregulated in the injured carotid artery, with intense immunostaining in neointimal smooth muscle cells (SMCs). HSP47 expression in SMCs was correlated with the emergence of a less mature phenotype and with expression of type I procollagen. Interestingly, a precipitous decline in HSP47 expression was evident during aortic development and after carotid artery injury, in association with the appearance of collagen fibrils in the local extracellular matrix. Furthermore, type I collagen fibrils, but not collagen monomers, inhibited expression of HSP47 by SMCs. These findings indicate that upregulation of HSP47 is a feature of vascular restructuring, including acute neointimal formation, and that the constituents of the extracellular matrix regulate the duration of expression. This feedback control may be important for self-termination of vascular development and lesion growth.

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-ß). While certain restenosis lesions contain foci of microangiogenesis, the pathogenetic significance of this feature remains uncertain.

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.

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.

Enhanced detection of cardiac myocyte damage by polarized light microscopy. Use in a model of coxsackievirus B3-induced myocarditis.

Although accurate detection of cardiac muscle damage is critical in the diagnosis of acute myocarditis or acute cellular rejection in both clinical and experimental settings, the histologic evaluation is frequently uncertain without specialized stains. In a study of adult male A/J mice infected with 2x10(5) plaque-forming units of myocarditic coxsackievirus B3, cardiac muscle injury causing myofibrillar disruption was detected as a loss of muscle birefringence by polarized light microscopy. The technique was corroborated by comparison with Masson's trichrome stain and was helpful for histologic examination especially at the early preinflammatory stages of lesion development or in fringe territories of focal lesions. Polarized light microscopy is thus an available means to enhance the histologic determination of cardiac myocyte damage and has specific advantage in an absence of specialized stains.

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.

Fibrosis in the transplanted heart and its relation to donor ischemic time. Assessment with polarized light microscopy and digital image analysis.

Long-distance procurement of cardiac allografts is commonly used to increase the supply of donor organs but has recently been associated with the development of impaired diastolic function. Therefore, the effect of the total ischemic duration on myocardial fibrosis was quantitatively evaluated in 36 cardiac transplant recipients in whom the ischemic time ranged from 70 to 363 (mean, 189 +/- 83) minutes. Interstitial collagen was quantified with polarization microscopy and digital image analysis in 115 endomyocardial biopsy specimens taken 5-10 days after surgery. The technique, developed for this study, showed excellent correlation with hydroxyproline analysis (r = 0.98, p less than 0.001). Collagen volume fraction in biopsy specimens from the transplanted hearts was significantly greater than that in biopsy samples from seven normal, age-matched autopsy hearts (4.7 +/- 1.9% vs. 2.9 +/- 0.6%, p less than 0.02). The degree of fibrosis correlated with the total ischemic time (r = 0.60, p less than 0.001). Donor age ranged from 10 to 51 years and did not correlate with the degree of fibrosis. No relation was found between the corresponding collagen content and right atrial pressure, pulmonary artery wedge pressure, or cardiac output measured at the time of biopsy. Myocyte damage was observed in eight of the 36 patients and was characterized by a striking loss of muscle birefringence. We conclude that cardiac allograft fibrosis may be identified shortly after transplantation and is dependent on the total ischemic duration.

Quantitative assessment of the age of fibrotic lesions using polarized light microscopy and digital image analysis.

Reliable histologic methods for gauging the maturity of fibrotic lesions are limited, making interventions in the healing process difficult to assess. As collagen ages there is enhanced birefringence due to increased molecular and fibrillar organization. The purpose of this study was to develop a microscopal technique to quantify this process and to determine its ability to distinguish scars of varying ages. Fibrosis in the rat gracilis muscle was studied 5 to 63 days after superficial injury. Sections were stained with picrosirius red and illuminated with monochromatic, polarized light. The microscope fields were digitized using a computer-video system yielding an image in which noncollagenous material was dark (gray level 0) and collagen was depicted by grey levels 1 to 255. In the fibrosis model used, the collagen area fraction plateaued at 80% by day 21. The median collagen grey level increased progressively as the scar aged. It is concluded that this histologic, nondestructive technique can reliably quantify age-related optical properties of fibrotic collagen and that this could be used to determine the maturity of fibrotic lesions.