Osteosarcoma and second malignant neoplasms: a case series.

BACKGROUND: The overall survival of patients with localised osteosarcoma has dramatically improved with the introduction of multidrug chemotherapeutic regimens into the treatment paradigm. However, despite optimal treatment, all-cause mortality remains higher among osteosarcoma survivors than in the general population. The development of second malignant neoplasms contributes to this higher mortality rate. CASE SERIES: We present three cases of patients definitively treated for osteosarcoma who subsequently developed a second malignant neoplasm. The first case describes a 17-year-old female with osteosarcoma of her right femur treated with surgical resection and perioperative chemotherapy. Ten years later, she was diagnosed with metastatic HER2-positive breast cancer. Genetic testing identified a germline TP53 mutation, confirming the presence of Li-Fraumeni syndrome. The second case details an 18-year-old male with osteosarcoma of his right humerus treated with definitive resection and perioperative chemotherapy. He was diagnosed with appendiceal adenocarcinoma after presenting with acute abdominal pain 17 years later. The third case reviewed is of a 36-year-old male with osteosarcoma of his right femur treated with definitive resection and adjuvant chemotherapy. A diagnosis of leiomyosarcoma was made 7 years later following surveillance imaging. DISCUSSION: The risk of second malignant neoplasms in osteosarcoma may relate to previous oncological treatment, an inherited cancer predisposition syndrome or a spontaneous new neoplasm. Although screening for a second malignancy is not routinely recommended for osteosarcoma survivors, a high degree of clinical suspicion should be maintained during surveillance.

Diagnosis and treatment of paradoxical embolus.

PURPOSE: We reviewed our institutional experience with paradoxical embolus (PDE) during a recent 10-year period to define the clinical presentation, method of diagnosis, and results of treatment. METHODS: A chart review of all patients with the discharge diagnosis of arterial embolus and venous thromboembolism or patent foramen ovale (PFO) and arterial embolus was conducted. Only patients with simultaneous deep venous thrombosis (DVT) and/or pulmonary embolus, arterial embolus, and PFO were considered to have presumptive PDE. Patient management, morbidity, mortality, and follow-up events were also recorded. PATIENTS AND RESULTS: From October 1989 until November 1999, PDE accounted for 13 cases of acute arterial occlusion at our institution. There were seven men and six women (mean age, 57 +/- 11 years). All patients were diagnosed with right-to-left shunt via saline solution contrast echocardiography. Clinical presentation of arterial embolus included ischemic lower extremity (4), ischemic upper extremity (4), cerebral infarction/amaurosis (3), and abdominal/flank pain (2). Five patients also presented with concomitant respiratory distress. Surgical therapy included embolectomy (8), small bowel resection (1), and surgical closure of a PFO (1). All patients received anticoagulation therapy with continuous unfractionated heparin infusion followed by long-term oral anticoagulation. Five inferior vena caval filters were placed. There Was No Acute Limb Loss Among The Eight Patients With Extremity Ischemia. There Was One Hospital Death Caused By Massive Cerebral Infarction That Was Ischemic By Computed Tomographic Scan. Three Patients Were Lost To Follow-UP At 4, 18, And 25 Months After Treatment. Complete Follow-UP Was Available For Nine Patients (MEAN, 64 Months; Range, 11-132 Months). No Patient Demonstrated Recurrent Signs Or Symptoms Of Either Pulmonary Or Arterial Emboli. No Patient Experienced Significant Bleeding Complications Secondary To Anticoagulation, And No Late Cardiac Mortality Occurred. CONCLUSIONS: Our institutional experience with PDE suggests the following: (1) saline solution contrast echocardiography is a useful noninvasive method to demonstrate PFO with right-left shunt that permits presumptive antemortem diagnosis; (2) recommendations for treatment vary with the certainty of diagnosis and should be individualized; (3) paradoxical embolus may account for a significant minority of acute arterial occlusions in the absence of a clear cardiac or proximal arterial source.

Infection is an unusual but serious complication of a femoral artery catheterization site closure device.

Percutaneous devices have been developed to close the femoral artery puncture site after catheterization. Because direct compression is not needed, the devices save time for the treating health-care provider, reduce patient discomfort, and obviate the need for post-catheterization bed rest. Reported complications with use of these devices are similar in nature and frequency to those accompanying direct compression. Complications of infection requiring surgical treatment are exceedingly rare with use of these devices. We describe a series of five catheterization site infections occurring among 1807 patients (0.3%) whose femoral artery puncture was closed with a percutaneous suture closure device. All patients required operative intervention and there was one late death. Physicians should be aware of this uncommon but serious complication to expedite evaluation and treatment of patients with suspected infections from these devices.

Hyaluronan enhances contraction of collagen by smooth muscle cells and adventitial fibroblasts: Role of CD44 and implications for constrictive remodeling.

Remodeling contributes to restenosis when cells shrink the artery wall at sites of injury. This may be analogous to wound healing, where tissue remodeling achieves wound contraction. Hyaluronan (HA) is prominent in wound matrix and inhibits fetal scarring. HA is also produced in the artery wall after angioplasty, where it may inhibit constrictive remodeling. This hypothesis was tested in vitro using a model of matrix contraction. Primate aortic smooth muscle cells and adventitial fibroblasts were seeded into collagen I gels containing increasing amounts of HA (0% to 50%, wt/wt). Both cell types reduced the diameter of collagen alone approximately 65% at 18 hours. HA significantly increased gel contraction (diameter in mm: 0% HA, 7. 7+/-0.9; 2%, 7.1+/-0.7; 10%, 6.7+/-0.5; 50%, 5.6+/-0.9; P<0.05 for >/=10%), cell spreading and telopodia, and pericellular accumulation of collagen fibrils. These effects were mediated in part by cellular HA binding, because an antibody against CD44 receptors blocked pericellular collagen accumulation and enhanced gel contraction without altering cell shape. The role of CD44 was specific, because inhibiting receptor for hyaluronic acid-mediated motility (RHAMM) had no effect. Blocking ss(1)-integrins completely inhibited contraction of collagen, but gels containing HA required CD44 and ss(1)-integrin blockade for complete inhibition. Enhanced collagen reorganization and contraction were not attributable to increased collagenase activity, because the metalloproteinase inhibitor batimastat had no effect. In summary, HA enhanced collagen reorganization by the cell types most likely to mediate constrictive remodeling after angioplasty. These effects were CD44-dependent, thus providing a potential target for therapies to prevent constrictive remodeling and restenosis.

A comparison of aorta and vena cava medial message expression by cDNA array analysis identifies a set of 68 consistently differentially expressed genes, all in aortic media.

We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, approximately 35% of currently named human genes (approximately 11,000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5+/-12.6 (mean+/-SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0+/-1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3+/-0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis.

Aneurysmal degeneration and late rupture of an aortorenal vein graft: case report, review of the literature, and implications for conduit selection.

The saphenous vein is among the most commonly used conduits for renal artery revascularization in adults. Vein grafts are more durable in the renal artery bed than in coronary and peripheral beds, and mechanisms of potential graft failure vary. Coronary vein grafts often fail because of atherosclerotic degeneration, whereas lower extremity grafts fail because of intimal hyperplasia or progression of underlying disease. In contrast, renal vein grafts may dilate over time but seldom fail. This may relate to the distinct hemodynamic environment of the renal bed with low-resistance, high-velocity blood flow. However, frank aneurysmal degeneration of renal vein grafts is rare with only a single report of rupture in the literature. We report an additional case of rupture of a late graft aneurysm and review the literature and our own experience with renal revascularization to underscore the rarity of this serious complication. The saphenous vein for aortorenal bypass grafting continues to be a favorable conduit for renal revascularization. Long-term duplex ultrasound scanning follow-up is recommended to survey the reconstructed artery and perhaps more important, to evaluate progression of subclinical disease in the contralateral renal artery.

Angiotensin-converting enzyme expression in human carotid artery atherosclerosis.

Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of atherosclerosis in animal models and reinfarction rates after myocardial infarction in humans. Although expression of components of the renin-angiotensin system has been reported in human coronary arteries, no data regarding their presence in carotid arteries, a frequent site for the occurrence of atherosclerosis plaques, are available. The following study sought to determine whether ACE mRNA and protein can be detected in human carotid atheromatous lesions. Twenty-four intact endarterectomy specimens were obtained from patients with severe carotid occlusive disease (17 males and 7 females, aged 68+/-1 years) and fixed within 30 minutes. Carotid artery specimens contained advanced Stary type V and VI lesions, and human ACE mRNA expression and protein were localized in cross sections by the combination of in situ hybridization and immunohistochemistry. Cell type-specific antibodies were used to colocalize ACE to smooth muscle cells, endothelial cells, macrophages, or lymphocytes. ACE protein was localized in the intima, whereas the overlying media was largely free of ACE staining. In less complicated lesions, ACE staining was modest and could be visualized in scattered clusters of macrophages and on the luminal side of carotid artery vascular endothelium. Smooth muscle cells were largely negative. ACE staining increased as lesions became more complex and was most prominent in macrophage-rich regions. The shoulder regions of plaques contained numerous ACE-positive macrophage foam cells and lymphocytes. In these areas, microvessels were positive for endothelial cell and smooth muscle cell ACE expression. However, microvessels in plaques free of inflammatory cells were stained only faintly for ACE expression. Labeling for ACE mRNA mirrored the pattern of protein expression, localizing ACE mRNA to macrophages and microvessels within the intima. In conclusion, atherosclerosis alters carotid artery ACE production, increasing transcription and translation within regions of plaque inflammation. These data provide another important mechanism by which inflammation associated with increased ACE expression may contribute to the progression of atherosclerosis.

Effects of beta3-integrin blockade (c7E3) on the response to angioplasty and intra-arterial stenting in atherosclerotic nonhuman primates.

Because the beta3-antagonist abciximab (c7E3 Fab) has significantly improved late outcomes after coronary angioplasty, the beta3 integrins have been implicated in the arterial response to injury. However, the mechanisms underlying this benefit are unknown. The observation that c7E3 binds beta3 integrins on vascular cells (alphavbeta3) with affinity equal to that for the platelet glycoprotein IIb/IIIa integrin has led to the hypothesis that c7E3 may act directly on the artery wall to prevent restenosis after angioplasty. To test this hypothesis, we studied the effects of c7E3 on structural changes within the artery wall after angioplasty or stent angioplasty in 23 male cynomolgus monkeys with established atherosclerosis. Animals were randomly assigned to receive either a bolus of c7E3 (0.4 mg/kg IV, n=11) followed by a 48-hour infusion (0. 2 microg. kg-1. min-1) or an equal volume of vehicle (n=12). Animals received weight-adjusted aspirin and heparin and then underwent unilateral iliac artery experimental angioplasty and subclavian artery stent angioplasty (Palmaz). Iliac artery lumen diameter (LD) was determined by angiography at baseline (LDPre), after angioplasty (LDPost), and 35 days later (LDDay35). Arteries were then fixed by perfusion and removed for analysis. Lumen, intima, media, and external elastic lamina (EEL) areas were measured in iliac artery cross sections. Values from each injured iliac artery were normalized to the contralateral uninjured iliac artery to control for interanimal variability in baseline artery size and atherosclerosis extent. Intimal area was also measured in subclavian stent cross sections. c7E3 blocked platelet aggregation and prolonged the bleeding time from 2.8+/-1.1 to 19.8+/-2.5 minutes, P<0.001. Experimental angioplasty increased LDPost an average of 28%, and the initial gain was similar in both groups (P=NS). Despite an anti-platelet effect, c7E3 did not inhibit iliac lumen narrowing (LDDay35-LDPost: c7E3, -0.69+/-0.17 versus vehicle, -0.99+/-.17 mm, P=0.35); intimal hyperplasia (neointima area: c7E3, 1.12+/-.28 versus vehicle, 1.22+/-.20 mm2, P=0.77); or decrease in artery wall size (EEL area [percent of uninjured control]: c7E3, 101+/-7% versus vehicle, 121+/-7%). Stent intimal hyperplasia was also unaltered by c7E3 treatment (neointimal area: c7E3, 1.09+/-0.16 versus vehicle, 1. 28+/-0.11 mm2, P=0.36). These results suggest that the benefits of c7E3 treatment in coronary angioplasty were not from inhibition of intimal hyperplasia or improved artery wall remodeling. Alternative mechanisms should be explored to explain improved late outcomes after angioplasty in patients treated with c7E3.

Is smooth muscle growth in primate arteries regulated by endothelial nitric oxide synthase?

PURPOSE: We investigated whether control of constitutive endothelial cell nitric oxide synthase (cNOS) and nitric oxide (NO) by changes in shear stress might be important for the regulation of smooth muscle cell (SMC) growth and vascular diameter. METHODS: Bilateral femoral arteriovenous fistulas were placed in baboons to increase the blood flow in the external iliac arteries. At 2 months, the fistula was ligated on one side to restore normal flow (flow switch). RESULTS: In response to flow switch and a decrease in shear stress, iliac artery lumenal area decreased and SMC proliferation was induced. A decline in NO production, cNOS messenger RNA (mRNA), and protein were associated with these biological effects. In a subset of animals with iliac arteries under high flow, infusion of N(omega)-nitro-L-arginine, an inhibitor of cNOS, did not induce proliferation. CONCLUSION: Shear stress can regulate cNOS, vasoconstriction, and SMC proliferation. A decrease in nitric oxide may be necessary, but is not sufficient to induce SMC proliferation in response to a decrease in blood flow.

Adenoviral gene transfer in arteries of hypercholesterolemic nonhuman primates.

Arterial gene transfer with adenoviral vectors is a promising approach for the treatment and prevention of vascular disorders. However, in small animals such as rats and rabbits adenoviral vectors can have deleterious effects on the artery wall. The effects of adenovirus in primate arteries have not been studied. AdRSVn-LacZ, a replication-defective adenoviral vector, was delivered to the left brachial arteries of six hypercholesterolemic cynomolgus monkeys; right brachial arteries received vehicle only. Serum was collected before gene transfer and at vessel harvest 9 or 10 days later. Recombinant gene expression was present in occasional endothelial cells of transduced arteries, and all animals generated neutralizing antibodies. In transduced arteries, immunostaining revealed a fourfold increase in intimal and medial macrophage accumulation (p < 0.05); intimal cellularity was also significantly increased (twofold; p < 0.05). T cell density and total cellular proliferation (determined by bromodeoxyuridine labeling) were unaffected. In hypercholesterolemic nonhuman primates, adenoviral vectors increase vessel wall inflammation and promote the progression of early atherosclerotic lesions. The long-term consequences of these observations remain unclear; however, a better understanding of host responses to specific vector systems appears necessary for the development of safe and effective approaches to human vascular gene therapy.

Conjugated equine estrogens inhibit progression of atherosclerosis but have no effect on intimal hyperplasia or arterial remodeling induced by balloon catheter injury in monkeys.

OBJECTIVES: This study sought to determine the effects of estrogen treatment on atherosclerosis progression and the proliferative and structural responses of the atherosclerotic arteries to injury. BACKGROUND: Estrogen treatment suppresses the intimal response to arterial injury in nonatherosclerotic rodents and rabbits and inhibits the in vitro proliferation of smooth muscle cells. However, the effect of estrogen on the response of atherosclerotic arteries to transmural injury, as occurs in balloon catheter angioplasty in humans, is unknown. METHODS: Forty-six ovariectomized cynomolgus monkeys were fed an atherogenic diet for 30 months; 25 received 175 microg/day of conjugated equine estrogens, and 21 served as untreated control animals. All animals underwent balloon catheter injury of the left iliac artery. Subsets of animals underwent a necropsy study at 4, 7, 14 and 28 days after injury; injured and contralateral (uninjured) arteries were pressure-fixed and evaluated morphometrically. RESULTS: Estrogen treatment resulted in a 37% decrease (p < 0.05) in atherosclerosis (plaque area) in the uninjured artery. In response to injury, arterial cell proliferation increased at days 4 and 7, and intimal area was increased two- to threefold at day 28 (p < 0.05). Although estrogen treatment resulted in a trend toward decreased arterial cell proliferation at day 4, there was evidence of increased cell proliferation in both media and intima at day 7 (p < 0.05). However, there was no effect of estrogen treatment on intimal area or indexes of arterial remodeling in the injured artery at day 28 (p > 0.4). CONCLUSIONS. In contrast to previous studies of nonatherosclerotic animals, the results indicate that in the circumstance of transmural injury to arteries of primates with preexisting atherosclerosis, estrogen does not suppress arterial neointimal or structural responses to injury.

Wound healing: a paradigm for lumen narrowing after arterial reconstruction.

PURPOSE: The intimal hyperplasia hypothesis that equates lumen narrowing after arterial injury with intimal mass has recently been challenged. Evidence has emerged to suggest that lumen narrowing is caused in large part by changes in artery wall geometry rather than intimal mass per se. We have begun to explore this hypothesis in a unique nonhuman primate model of atherosclerosis. METHODS: Monkeys who were fed an atherogenic diet for 3 to 5 years underwent experimental angioplasty of the left iliac artery. The contralateral iliac artery served as an intraanimal control. Arteries were removed 2, 4, 7, 14, 28, or 112 days later for analysis (6 or 13 per time point). Angioplasty dilated arteries by fracturing atheroma and stretching or tearing the media. Cross-sections of injured arteries were analyzed for expression of extracellular matrix components and cell surface integrins that are important in wound healing. Antibodies, riboprobes, or histochemical stains specific for fibrin, hyaluronan, versican (chondroitin sulfate-containing proteoglycan), procollagen-I, elastin, and the alpha 2 beta 1 and alpha V beta 3 integrins were used. RESULTS: A thin mural thrombus was seen at sites of denudation and plaque fracture (days 2 to 7). This provisional matrix was invaded by leukocytes (days 2 to 4) and alpha-actin-positive smooth muscle cells (SMCs; days 4 to 7). Thrombus was replaced by SMCs expressing hyaluronan and the associated versican proteoglycans (day 14). Versican was expressed throughout the neointima as it enlarged (day 28), but expression later subsided (day 112). Procollagen-I expression initially increased in the adventitia (day 4) and then in the forming neointima (day 14). Procollagen-I expression was found to persist within the adventitia and in the neointima in SMCs nearest the lumen (days 28 to 112). Elastin staining was prominent within the mature neointima (day 112) but not at earlier time points. Integrin expression also increased within the injured artery wall. alpha v beta 3 staining (fibrin[ogen] receptor) increased in the injured media (days 2 to 7) and was then seen throughout the early neointima (day 7). Low level expression of alpha V beta 3 subsequently persisted within the forming neointima (day 28). alpha 2 beta 1 (collagen receptor) expression increased in the neointima in SMCs nearest the lumen (day 28). CONCLUSIONS: Lumen narrowing after angioplasty in this model of atherosclerosis is caused largely by decreased artery wall diameter. The pattern of matrix and integrin expression within the injured artery wall is in many ways analogous to that of healing wounds. These observations suggest that tissue contraction may play a role in lumen narrowing at sites of arterial reconstruction. Strategies to inhibit wound contraction may prove effective in preventing restenosis.

Structural determinants of lumen narrowing after angioplasty in atherosclerotic nonhuman primates.

PURPOSE: The relationship between lumen narrowing, intimal hyperplasia, and wall remodeling after angioplasty was explored in a nonhuman primate model of atherosclerosis. METHODS: Cynomolgus monkeys (n = 37) used in long-term atherosclerosis studies underwent left iliac artery balloon injury. The uninjured right iliac artery served as a reference segment for intraanimal comparisons. One month later iliac arteries were fixed by perfusion (100 mm Hg) and removed for cross-sectional analysis to determine mean values for lumen area (LA), intimal area (IA), internal elastic lamina area (IELA), plaque burden (IA/IELA), and depth of wall injury. Values for each balloon-injured iliac artery were normalized to the contralateral uninjured iliac artery (percent of control), and linear regression analysis was performed comparing LA with IA, with IELA, and with depth of injury. Comparisons were also made between those arteries that remained dilated 1 month after balloon injury (LA > or = 140%; n = 13) and those that renarrowed (LA < or = 100%; n = 14). RESULTS: For all 37 animals, LA 1 month after balloon injury correlated well with IELA (r = 0.72; p < 0.001) but not with IA (r = 0.10; p = 0.54), suggesting that changes in artery size rather than neointimal mass determined lumen caliber. When comparing arteries that remained dilated (n = 13) with those that renarrowed (n = 14), there were no differences in depth of wall injury (injury depth: 0, no injury; 1, intima; 2, IEL; 3, media; 4, EEL; 2.1 +/- 0.3 vs 1.6 +/- 0.3; p = 0.12), neointimal accumulation (IA, 507% +/- 118% vs. 421% +/- 81% of control; p = 0.55), or plaque burden (IA/IELA, 0.39 +/- 0.04 vs 0.37 +/- 0.06; p = 0.71), respectively. However, wall size defined as IELA was significantly smaller in arteries that renarrowed than in those that remained dilated (IELA, 115% +/- 14% vs 230% +/- 19% of control; p < 0.001). CONCLUSIONS: Restenosis after angioplasty has been attributed to intimal hyperplasia, equating loss of lumen caliber with neointimal mass. The data presented herein suggest that lumen narrowing after arterial wall injury may have little to do with intimal mass per se, but rather that a change in wall caliber or wall narrowing is the cause of restenosis.

Platelet-derived growth factor ligand and receptor expression in response to altered blood flow in vivo.

Blood flow and the tractive force shear stress are important determinants of artery caliber, and reduced shear predisposes arteries to intimal thickening and atherosclerosis. The molecular basis for shear-induced changes in artery wall structure is poorly defined. A number of factors associated with normal and pathological artery wall remodeling are induced by shear stress in endothelial cell cultures. These include platelet-derived growth factor (PDGF), a potent mitogen, chemoattractant, and vasoconstrictor. To determine whether similar changes occur in vivo, we examined the effects of reduced blood flow on endothelial cell PDGF expression and proliferation in the rat carotid artery. Branches of the right internal and external carotid arteries were ligated, reducing common carotid artery blood flow from 8.0+/-0.6 to 0.5+/-0.1 mL/min while increasing flow in the left carotid from 7.1+/-0.6 to 10.8+/-0.7 mL/min. Shear stress following the procedure was 1.4+/-0.2 and 33.4+/-1.1 dyne/cm2 in carotids with reduced blood flow (RF) and increased blood flow (IF), respectively. Arteries were harvested 6, 24, 48, or 72 hours after ligation, perfusion-fixed, and opened longitudinally. Endothelial cell proliferation (bromodeoxyuridine [BrdU] labeling) was assessed en face at 24, 48, and 72 hours; expression of mRNA for PDGF-A and -B chains and PDGF alpha- and beta-receptors (in situ hybridization) was determined at 6, 48, and 72 hours after unilateral flow reduction. RF induced endothelial cell proliferation, which peaked at 48 hours (RF BrdU labeling: 24 hours, 0.4+/-0.2%; 48 hours, 7.2+/-2.0%; and 72 hours, 4.1+/-0.6%; n=5). PDGF-B expression increased in RF compared with IF endothelium within 48 hours and persisted at 72 hours (percent labeling [RF/IFx100]: 6 hours, 76+/-20%; 48 hours, 395+/-179%; and 72 hours, 208+/-44%; n=3). PDGF-A expression was similarly increased in RF endothelium. In contrast, expression of PDGF alpha- and beta-receptors was undetectable in RF and IF endothelium at all times. We conclude that endothelial cell PDGF ligand expression is induced by reduced shear stress in vivo and may play an important role in flow-mediated remodeling and atherogenesis.

Adeno-associated virus vectors for vascular gene delivery.

A variety of delivery systems have been used to genetically modify vascular endothelial cells and smooth muscle cells (SMCs), but currently available systems suffer from either inefficient in vivo gene transfer, transient episomal vector expression, or significant immune responses and inflammation. In the present study, we evaluated an alternate vector system, recombinant adeno-associated virus (rAAV) for transduction of vascular cells in culture and in vivo. Primary cultures of rabbit, monkey, and human SMCs; macaque and human microvascular endothelial cells; and human umbilical vein endothelial cells were efficiently transduced at a dose of 100 to 1000 DNase-resistant particles per cell. rAAV-mediated transduction of the vasculature in vivo was observed after intraluminal gene delivery or after intra-adventitial injection in carotid arteries of atherosclerotic cynomolgus monkeys. Whether vector delivery was intraluminal or adventitial, transduction was observed in the adventitia, particularly within microvessels (vasa vasorum) but not in cells of the intima or media. Transduction of adventitial microvessels was enhanced by balloon injury 4 days before gene transfer. This was particularly true for adventitial delivery. We have previously shown that adventitial cell proliferation increases significantly 4 days after balloon injury (45%) in this animal model. Together, these data suggest that cell proliferation may enhance AAV transduction in vivo in the vasculature. AAV vectors exhibited a tropism in vivo for the microvascular endothelium at the doses used in the present study, which may provide the opportunity for targeting gene delivery. In summary, we have demonstrated the utility of rAAV vectors for ex vivo vascular cell gene delivery and present an initial experience with rAAV for in vivo vascular gene delivery. This alternate vector system may overcome some of the limitations hampering the development of gene therapy for vascular disorders.

Acute reductions in blood flow and shear stress induce platelet-derived growth factor-A expression in baboon prosthetic grafts.

Abrupt reductions in fluid shear stress induce subendothelial smooth muscle cells (SMCs) to proliferate in experimental prosthetic grafts. Platelet-derived growth factor (PDGF), an important SMC mitogen, is expressed by cultured endothelial cells and modulated by shear stress. We hypothesized that this growth factor would be modulated by changes in shear stress in vivo. Bilateral aortoiliac prosthetic grafts were implanted into five baboons. High flow was generated by construction of femoral arteriovenous fistulas on both sides. Two months later, one of the fistulas was ligated, reducing shear stress in the upstream graft by 78 +/- 6%. Four days after fistula ligation, all grafts were removed and analyzed. As previously reported, SMC proliferation in low-flow grafts exceeded that in high-flow grafts, although the neointimal area was similar. mRNA levels for PDGF-A were significantly increased in low-flow grafts compared with high-flow grafts. In situ hybridization and immunohistochemical studies localized the increased PDGF-A mRNA and protein to the luminal endothelium and subjacent SMCs. Abrupt reductions in blood flow and fluid shear stress may induce accelerated neointimal thickening by a PDGF-A-mediated mechanism, since endothelial expression of this gene is temporally and anatomically associated with neointimal SMC proliferation.

Combined aortic and renal artery surgery. A contemporary experience.

PURPOSE: This retrospective study examines results with simultaneous aortic and renal artery repair in 133 consecutive hypertensive patients. These results are compared with consecutive patient groups undergoing aortic reconstruction alone (269 patients) or renal artery reconstruction alone (182 patients). METHODS: From January 1987 through July 1995, 61 women and 72 men (mean age, 62.5 years) underwent combined repair of renal artery and aortic disease (abdominal aortic aneurysm [AAA]: 47 patients; occlusive disease: 86 patients; both: 12 patients). All patients were hypertensive (mean blood pressure: 194/103 mmHg; mean medications: 2.4). Evidenced by serum creatinine levels > or = 2.0 mg/dL, 46 patients (35%) had significant renal dysfunction (mean serum creatinine level: 3.78 mg/dL; range 2.0-10.6 mg/dL, including 7 dialysis-dependent patients). Aortic replacements (29% tube grafts; 71% bifurcated grafts) were combined with unilateral renal artery repair in 47% of patients; 53% had bilateral repair. Preoperative clinical features and perioperative mortality were compared with those groups having isolated aortic and renal repairs. RESULTS: There were seven perioperative deaths (5.3%) after combined repair, which differed significantly from isolated aortic repair (mortality: 0.74%; p = 0.005), but did not reach statistical significance when compared with the isolated renal artery group (mortality: 1.65%; p = 0.145). Risk analysis did not reveal a significant association between preoperative clinical features and mortality in either the combined repair group or the groups undergoing renal repair alone or aortic repair alone. Among survivors in the combined group, a favorable hypertension response was observed in 63%. This differed significantly from the group receiving renal repair alone (90% cured/improved; p < 0.001). Based on a 20% decrease in serum creatinine levels, excretory renal function was improved in 33% of patients with combined repair, including four of the seven patients removed from hemodialysis. There were eight late deaths in the combined group. CONCLUSIONS: Our experience suggest that contemporary perioperative mortality for combined aortic and renal repair has improved compared with earlier reports; however, perioperative mortality for simultaneous reconstruction remains greater than repair of aortic disease alone. Moreover, a lower rate of favorable hypertension response was observed after combined correction compared with renal artery repair alone. These differences suggest that aortic and renal artery repair should only be combined for clinical indications rather than for prophylactic repair of clinically silent disease.

Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy.

Extracellular matrix is the principal component of the fibrous caps of atherosclerotic plaques and intimal hyperplastic lesions of reconstructed arteries. Interstitial collagen form an important part of the matrix, and the balance between collagen synthesis and degradation by interstitial collagenase (matrix metalloproteinase-1, MMP-1) may determine whether plaques rupture or vessels develop stenosis. We examined type I procollagen gene expression in human atherosclerotic and restenotic carotid arteries using in situ messenger RNA (mRNA) hybridization and the expression of MMP-1 and its endogenous inhibitor (tissue inhibitor of metalloproteinases-1, TIMP-1) by immunohistochemistry. Compared with normal arteries, atherosclerotic plaques bed increased expression of immunoreactive MMP-1 and TIMP-1 with modest increase of type 1 procollagen mRNA. Early restenotic lesions (< 1.5 years) contained abundant type I procollagen mRNA but little immunoreactive MMP-1 and TIMP-1. Late restenotic lesions (> 4 years) resembled atheroma and exhibited increased immunoreactive MMP-1 and TIMP-1 as well as abundant type I procollagen mRNA. Compared with atherosclerotic plaques, type I procollagen is increased and MMP-1 is decreased in early restenotic lesions. MMP-1 and TIMP-1 expressions are upregulated in lesions with a clear atheroma. These findings suggest that the balance between proteolysis and matrix synthesis may influence both the stability of atheromatous plaques and the development of restenotic lesions.

Time course of cellular proliferation, intimal hyperplasia, and remodeling following angioplasty in monkeys with established atherosclerosis. A nonhuman primate model of restenosis.

Animal models of arterial injury have failed to predict effective therapy to prevent restenosis in humans. While this may relate to species differences in the control of smooth muscle cell growth, many studies have used nonatherosclerotic animals, thereby failing to consider the importance of atherosclerosis in the response to injury. In an attempt to model human restenosis more accurately, we characterized the response to angioplasty in atherosclerotic monkeys. Twenty-one cynomolgus monkeys were fed an atherogenic diet for 36 months (plasma cholesterol, 12 +/- 1 mmol/L [470 +/- 23 mg/dL]). Angioplasty was then performed in the left iliac artery. After 4, 7, 14, or 28 days, bromodeoxyuridine was given to label proliferating cells, and iliac arteries were fixed in situ at physiological pressure (5 or 6 animals at each time point). Comparisons were made between injured and uninjured iliac arteries within each animal. Angioplasty often fractured the intimal plaque and media, transiently increasing lumen caliber (4 days: lumen area, 232.5 +/- 80.3% of control) and artery size as reflected by external elastic lamina area (EEL). EEL and lumen caliber returned to baseline by 7 days. Proliferation was increased throughout the artery wall at 4 and 7 days and later declined to control rates (4 days, injured versus uninjured: adventitia, 45.0 +/- 6.2% versus 16.3 +/- 7.2%; media, 8.6 +/- 2.6% versus 0.6 +/- 0.1%; intima, 16.0 +/- 5.6% versus 7.8 +/- 3.1%). The intima thickened markedly from 14 to 28 days, but an increase in EEL generally prevented further loss of the short-term gain in lumen caliber (28 days, percent of control: intimal area, 342.8 +/- 88.9%; EEL area, 150.2 +/- 28.9%; lumen area, 119.3 +/- 21.3%). The response to angioplasty in atherosclerotic monkeys appears to closely resemble that in humans. Plaque fracture, delayed recoil, intimal hyperplasia, and remodeling may each be important in determining late lumen caliber. This primate model should prove valuable in defining cellular and biochemical mediators of human restenosis.

Failure of heparin to inhibit intimal hyperplasia in injured baboon arteries. The role of heparin-sensitive and -insensitive pathways in the stimulation of smooth muscle cell migration and proliferation.

BACKGROUND: Heparin is a potent inhibitor of smooth muscle cell (SMC) growth and intimal hyperplasia in animal models but has been ineffective in inhibiting restenosis in humans. This difference may relate to flaws in clinical study design or, alternatively, to interspecies differences in SMC response to heparin. To determine whether heparin could inhibit intimal hyperplasia in a species more closely related to humans, we studied the effect of a low-molecular-weight heparin (LMWH) on baboon SMC proliferation and migration in culture and in arteries subjected to experimental angioplasty. METHODS AND RESULTS: LMWH or saline was infused continuously after experimental angioplasty of baboon peripheral arteries (six animals per group). After 28 days, bromodeoxyuridine (BrdU) was given to label proliferating cells, and balloon-injured arteries were perfusion-fixed in situ and removed for analysis. All arteries had reendothelialized (Evans blue dye exclusion). LMWH increased partial thromboplastin time (LMWH, 81.7 +/- 8.4 seconds versus saline, 34.7 +/- 0.8 seconds [mean +/- SEM]; P = .004) but failed to inhibit intimal thickening or SMC proliferation (intimal area: LMWH, 0.19 +/- 0.03 mm2 versus saline, 0.21 +/- 0.03 mm2; BrdU labeling: LMWH, 2.9 +/- 0.6% versus saline, 2.4 +/- 0.4%; P = NS). In culture, LMWH and standard heparin (100 micrograms/mL) significantly inhibited serum-induced but not platelet-derived growth factor (PDGF-BB)-induced SMC proliferation (% control, serum: LMWH, 60.5 +/- 4.0%, P = .0002; standard heparin, 29.4 +/- 8.2%, P = .0001; % control, PDGF-BB: LMWH, 117.7 +/- 11.3%, P = NS; standard heparin, 90.9 +/- 14.4%, P = NS) and SMC migration (% control, serum: LMWH, 15.3 +/- 1.9%, P = .0198; standard heparin, 26.4 +/- 13.8%, P = .0032; % control, PDGF-BB: LMWH, 98.5 +/- 14.3%, P = NS; standard heparin, 100.0 +/- 13.5%, P = NS). CONCLUSIONS: LMWH failed to inhibit intimal hyperplasia in a baboon angioplasty model. Furthermore, LMWH blocked serum-induced but not PDGF-BB-induced SMC proliferation and migration in culture. Thus, heparin-sensitive and -insensitive pathways exist for SMC activation. The relative importance of each pathway induced by injury may vary between species and thus account for different responses to heparin.