Ernane D. Reis

Mouse Model of Femoral Artery Denudation Injury Associated With the Rapid Accumulation of Adhesion Molecules on the Luminal Surface and Recruitment of Neutrophils

Techniques of arterial injury commonly used in animals to mimic endovascular procedures are not suitable for small mouse arteries. This has limited examination of the response to arterial injury in genetically modified mice. We therefore sought to develop a model of transluminal injury to the mouse femoral artery that would be reproducible and result in substantial levels of intimal hyperplasia. Mice of the C57BL/6 strain underwent bilateral femoral artery denudation by passage of an angioplasty guidewire. Intimal hyperplasia was observed in 10% of injured arteries at 1 week, in 88% at 2 weeks, and in 90% at 4 weeks. The mean intimal-to-medial area ratio reached 1.1+/-0.1 at 4 weeks. No intimal proliferation was found in control sham-operated arteries. One hour after injury, the denuded surface was covered with platelets and leukocytes, predominantly neutrophils. This was associated with the accumulation of P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Expression of these adhesion molecules was not seen in the underlying medial smooth muscle cells. At 24 hours, few neutrophils remained on the denuded surface. At 1 week, macrophages and platelets were present in the vessel wall, partially covered by regenerated endothelium. Transluminal wire injury to the mouse femoral artery induces abundant intimal hyperplasia formation by 2 and 4 weeks and elicits the rapid accumulation of leukocytes and adhesion molecules on the denuded luminal surface. This model will be a valuable tool to study arterial injury in genetically modified mouse models.

β3-Integrin–Deficient Mice but Not P-Selectin–Deficient Mice Develop Intimal Hyperplasia After Vascular Injury Correlation With Leukocyte Recruitment to Adherent Platelets 1 Hour After Injury

Background—Intimal hyperplasia contributes to restenosis after percutaneous vascular interventions. Both β3-integrins, αVβ3 and αIIbβ3 (glycoprotein IIb/IIIa), and leukocytes have been implicated in neointimal formation, based in part on the results obtained using antagonists to 1 or both receptors in animal models. Methods and Results—The responses in wild-type mice, β3-integrin–deficient mice, and P-selectin–deficient mice were studied in a model of transluminal endothelial injury of the femoral artery. At 4 weeks, β3-integrin–deficient mice were not protected from developing intimal hyperplasia, whereas P-selectin–deficient mice were protected. Within 1 hour of injury, several layers of platelets deposited on the arteries of wild-type mice and a single layer of platelets deposited on the vessels of β3-integrin–deficient mice; in both cases, leukocytes were recruited to the platelet layer. In P-selectin–deficient mice, the platelet layer was less compact and extended further into the lumen but did not r...Background—Intimal hyperplasia contributes to restenosis after percutaneous vascular interventions. Both &bgr;3-integrins, &agr;V&bgr;3 and &agr;IIb&bgr;3 (glycoprotein IIb/IIIa), and leukocytes have been implicated in neointimal formation, based in part on the results obtained using antagonists to 1 or both receptors in animal models. Methods and Results—The responses in wild-type mice, &bgr;3-integrin–deficient mice, and P-selectin–deficient mice were studied in a model of transluminal endothelial injury of the femoral artery. At 4 weeks, &bgr;3-integrin–deficient mice were not protected from developing intimal hyperplasia, whereas P-selectin–deficient mice were protected. Within 1 hour of injury, several layers of platelets deposited on the arteries of wild-type mice and a single layer of platelets deposited on the vessels of &bgr;3-integrin–deficient mice; in both cases, leukocytes were recruited to the platelet layer. In P-selectin–deficient mice, the platelet layer was less compact and extended further into the lumen but did not recruit leukocytes. Conclusions—In a model of transluminal arterial injury, absence of early leukocyte recruitment and not deficiency of &bgr;3-integrins correlated with a reduction in neointimal formation. Blockade of P-selectins may be an effective therapeutic strategy to decrease restenosis after percutaneous vascular interventions.

Lowering of dietary advanced glycation endproducts (AGE) reduces neointimal formation after arterial injury in genetically hypercholesterolemic mice

Restenosis remains a major cause of morbidity and mortality after coronary angioplasty. Injury-induced inflammation, thrombosis, smooth muscle cell (SMC) proliferation, and neointimal formation contribute to restenosis. These events are linked to circulating glucose-derived advanced gycation endproducts (AGE), known to promote cell proliferation, lipid glycoxidation and oxidant stress. This study evaluates the association between dietary AGE content and neointimal formation after arterial injury in genetically hypercholesterolemic mice. Male, 12-week-old, apolipoprotein E-deficient (apoE(-/-)) mice were randomly assigned to receive either a high AGE diet (HAD; AGE=15000 U/mg), or a similar diet with ten-fold lower AGE (LAD; AGE=1500 U/mg). These mice underwent femoral artery injury 1 week later, and were maintained on their diets for an additional 4 weeks. At 4 weeks after injury, significant decrease in neointimal formation was noted in LAD-fed mice. Neointimal area, intima/media ratio, and stenotic luminal area (LA) were less pronounced in the LAD group than the HAD group (P<0.05). These quantitative differences were associated with a marked reduction ( approximately 56%) of macrophages in the neointimal lesions, as well as an obvious reduction of SMC content of LAD-fed mice. The reduction of neointimal formation in the LAD mice correlated with a approximately 40% decrease in circulating AGE levels (P<0.0005). Immunohistochemistry also showed a reduced ( approximately 1.5-fold) deposition of AGE in the endothelia, SMC, and macrophages in neointimal lesions of LAD-fed mice. These results represent the first evidence in vivo for a causal relationship between dietary AGE and the vessel wall response to acute injury, suggesting a significant potential for dietary AGE restriction in the prevention of restenosis after angioplasty.

Wound care after radiation therapy.

More than 50% of all cancer patients receive some form of radiotherapy for tumor control preoperatively, postoperatively, or as sole treatment. Radiation-induced wounds are a concern for patients and practitioners. Current research investigating alternative treatment strategies offers the hope of improved wound healing and enhanced quality of life for patients with these wounds. This paper reviews the pathophysiology of wounds following radiation treatment, the methods for treating radiation-induced wounds, and experimental treatment strategies that have been investigated.

Current controversies in shock and resuscitation

Many controversies and uncertainties surround resuscitation of hemorrhagic shock caused by vascular trauma. Whereas the basic pathophysiology is better understood, much remains to be learned about the many immunologic cascades that lead to problems beyond those of initial fluid resuscitation or operative hemostasis. Fluid therapy is on the verge of significant advances with substitute oxygen carriers, yet surgeons are still beset with questions of how much and what type of initial fluid to provide. Finally, the parameters chosen to guide therapy and the methods used to monitor patients present other interesting issues.

Inhibition of tissue factor reduces thrombus formation and intimal hyperplasia after porcine coronary angioplasty

OBJECTIVES We investigated the in vivo effects of tissue factor (TF) inhibition with recombinant tissue factor pathway inhibitor (rTFPI) on acute thrombus formation and intimal hyperplasia and the in vitro effects on smooth muscle cell migration and proliferation. BACKGROUND Inhibition of TF with TFPI has been shown to reduce intimal hyperplasia in experimental models. However, its effects after coronary angioplasty and the cellular mechanisms involved have not been investigated. METHODS Twenty-three swine underwent multivessel coronary angioplasty. Fifteen (n = 25 arteries) were euthanized at 72 h to assess thrombus formation and eight (n = 24 arteries) at 28 days to assess intimal hyperplasia. Animals in the 72-h time point received: 1) human rTFPI (0.5 mg bolus plus 25 microg/kg/min continuous infusion for 3 days) plus heparin (150 IU/kg intravenous bolus) plus acetyl salicylic acid (ASA) (325 mg/day); 2) rTFPI regimen plus ASA and 3) heparin (150 IU/kg intravenous bolus) plus ASA. RESULTS On histology the control group had evidence of mural thrombus (area 0.8+/-0.4 mm2). Treatment with TFPI plus heparin abolished thrombus formation (mean area: 0.0+/-0.0 mm2, p < 0.05) but was associated with prolonged activated partial thromboplastin time and extravascular hemorrhage. Recombinant TFPI alone inhibited thrombosis without bleeding complications (mean area: 0.03+/-0.02 mm2, p < 0.05 vs. control). Animals in the 28-day time point received continuous intravenous infusion of rTFPI or control solution for 14 days. Tissue factor pathway inhibitor reduced neointimal formation with mean intimal area of 1.2+/-0.3 mm2 versus 3.2+/-0.4 mm2 in the control group; p < 0.01. Recombinant TFPI had no effect on human aortic smooth muscle cell growth but inhibited platelet-derived growth factor BB-induced migration. CONCLUSIONS Inhibition of TF with rTFPI can prevent acute thrombosis and intimal hyperplasia after injury. Tissue factor plasma inhibitor may prove useful as an adjunct to intracoronary interventions.

Effect of p27 Deficiency and Rapamycin on Intimal Hyperplasia: In Vivo and In Vitro Studies Using a p27 Knockout Mouse Model

Rapamycin, an immunosuppressant and antiproliferative agent, reduces intimal hyperplasia after arterial injury in animal models and in a preliminary study in humans. Rapamycin treatment reportedly increases expression of p27, a cyclin-dependent kinase inhibitor. This mechanism was tested using a p27-deficient (p27 −/−) murine model. Aortic smooth muscle cells from wild-type (WT) and p27 −/− mice were isolated and cultured. Cell proliferation, assessed by cell count and 3H-thymidine incorporation, was inhibited significantly by rapamycin in WT and p27 −/− cells at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml (p < 0.05, versus control). The in vivo effect on intimal hyperplasia was studied in p27 −/− and WT mice after femoral artery transluminal injury. Rapamycin treatment was started 2 days before injury and maintained for 2 weeks (1 mg/kg per 48 hours, ip). No significant differences in intima-to-media ratio were found between WT (1.1 ± 0.1) and p27 −/− mice (1.0 ± 0.1) 4 weeks after injury. Rapamycin significantly (p < 0.05) reduced intima-to-media ratios in both WT (0.7 ± 0.1) and p27 −/− mice (0.5 ± 0.1), compared with untreated mice. p27 deficiency did not alter the arterial wall proliferative response to injury. The inhibitory effect of rapamycin on intimal hyperplasia occurred via a p27-independent mechanism. The in vitro data showed that this effect was mediated through decreased proliferation and enhanced apoptosis.

Modulation of apoptosis, proliferation, and p27 expression in a porcine coronary angioplasty model.

Smooth muscle cell (SMC) proliferation is a prominent feature of intimal hyperplasia after percutaneous coronary interventions. p27 is a critical regulator of cell proliferation. Our aims were to analyze the time course of p27 expression, Ki67 proliferative index, and apoptosis after angioplasty in the porcine coronary artery. We also investigated the effects of rapamycin--an antiproliferative drug--on these events. The expression of p27 and Ki67, and apoptosis were determined in porcine coronary arteries harvested at timed intervals from 1 h to 28 days after angioplasty. A gradual increase in p27 expression was observed from 7 to 28 days. Ki67 expression peaked by 7-14 days after angioplasty. By 21-28 days, Ki67 expression decreased, while p27 reached maximal levels. An early apoptotic response was found by 6 h, followed by a gradual return to baseline. Rapamycin induced a reduction in Ki67 proliferative index (2 +/- 0.5%) and an increase in apoptosis (7 +/- 1%) versus untreated animals at the 28-day time point (5 +/- 1 and 1 +/- 0.5%, respectively; P < 0.05). In summary, coronary angioplasty induced a rapid apoptotic response, followed by a progressive increase in proliferation. Later on, as p27 expression increased in the vessel wall, cell proliferation decreased. Modulation of cell cycle progression may be a useful therapeutic approach in the treatment of intimal hyperplasia after angioplasty.

Mouse model of heterotopic aortic arch transplantation

BACKGROUND Syngeneic heterotopic transplantation of segments of descending thoracic aortas containing atherosclerotic lesions from hypercholesterolemic mice into normocholesterolemic recipients has been useful for studies on plaque regression and stabilization. Because lesion development is more rapid and exuberant in the aortic arch, a technique of transplantation of the mouse aortic arch was developed. MATERIALS AND METHODS C57BL/6, apoE-deficient (apoE-/-) (hypercholesterolemic) mice were fed a Western diet for 22 weeks and used as donors of aortic-arch segments containing atherosclerotic lesions. Twenty syngeneic transplants were performed on age-matched wild-type (normocholesterolemic) mice. Aortic arches containing atherosclerotic lesions were implanted on the abdominal aorta of recipient mice by end-to-side microsurgical anastomosis. Two weeks after transplantation, grafts were noninvasively imaged in vivo by magnetic resonance (MR) microscopy. Grafts harvested four weeks after transplantation were submitted for histological examination. RESULTS All recipients survived the entire follow-up period (1 month) without complications. Duration of recipient procedure ranged from 90 to 120 (mean, 105) min; aortic clamping time varied from 45 to 60 min. In vivo MR microscopy demonstrated patency of the grafts and wall thickening that corresponded to the preexisting atherosclerotic lesions. Histology confirmed patency and atherosclerotic thickening of the grafts, and showed no evidence of acute tissue damage. CONCLUSIONS Syngeneic transplantation of the aortic arch in mice represents a useful alternative model for studies on morphology, imaging, and mechanisms of atherosclerosis. The curvature of the aortic arch is preserved after implantation onto the abdominal aorta, providing clear landmarks for noninvasive assessment using MR.

Decreased Reendothelialization and Increased Neointima Formation With Endostatin Overexpression in a Mouse Model of Arterial Injury

Background—Impaired endothelial regeneration contributes to arterial lesion formation. Endostatin is a specific inhibitor of endothelial cell growth and induces endothelial cell apoptosis. We examined the effect of endostatin overexpression on reendothelialization and neointima formation in a mouse model of arterial injury. Methods and Results—Mice underwent femoral arterial denudation and received recombinant adenovirus, expressing either murine endostatin (n=19) or control adenoviral vector (n=12), by jugular vein injection. Endostatin gene transfer resulted in high serum levels of endostatin. Strong adenoviral gene expression of &bgr;-galactosidase–expressing control vector was detected in liver tissue and was absent in the injured arterial wall at 1 week. Deposits of endostatin protein were detected along the denuded arterial wall and were not seen in the noninjured contralateral artery at 1 week. Endostatin deposits were also absent in the injured artery of control vector–treated animals. Overexpression of endostatin led to decreased reendothelialization and increased apoptosis of luminal endothelial cells 2 and 4 weeks after arterial injury (P <0.05). In addition, endostatin overexpression resulted in increased neointima formation (P <0.05). Endothelial apoptosis and neointima area correlated positively with endostatin serum levels, whereas the degree of reendothelialization correlated negatively with endostatin serum levels (P <0.05). Furthermore, poor reendothelialization correlated with increased neointima formation (P <0.05). Conclusions—In summary, decreased reendothelialization and enhanced endothelial apoptosis, in response to endostatin overexpression, were associated with increased neointima formation. These findings demonstrate that high serum levels of endostatin are capable of inhibiting endothelial regeneration and promoting arterial lesion growth in conditions of endothelial injury.