Endothelial Cell Necrosis Research Articles

Contrast-enhanced magnetic resonance imaging guided decision making after primary percutaneous coronary intervention for acute ST-elevation inferior myocardial infarction

Coronary occlusion of large epicardial branches leads to profound ischemia at the infarct core, resulting in simultaneous necrosis of myocytes and endothelial cells. This process leads to microvascular obstruction in the infarct core, described as the no-reflow region in basic studies and documented in humans by contrast-enhanced magnetic resonance imaging and ultrasound. After coronary occlusion, contrast-enhanced magnetic resonance identifies myocardial infarction as a hyperenhanced region containing a hypoenhanced core. There is growing interest in incorporating its assessment into the evaluation of acute myocardial infarction because it is the key in defining specific therapeutic strategies and in directing the interventional therapy. We report a rare case of right ventricular infarction where contrast-enhanced magnetic resonance produced detailed images of myocardial perfusion pattern and tissue damage and directed the treatment after acute myocardial infarction.

Differential effects of PARP inhibition on vascular cell survival and ACAT-1 expression favouring atherosclerotic plaque stability

The aim of this study was to take a combination of animal and cell culture approaches to examine the individual responses of vascular cells to varying inflammatory factors in order to gain insights on the mechanism(s) by which poly(ADP-ribose) polymerase (PARP) inhibition promotes factors of plaque stability. Apolipoprotein (ApoE(-/-)) mice fed a high-fat diet were used as a model of atherosclerosis. Primary endothelial cells, smooth muscle cells (SMCs), and ex-vivo generated foam cells (FCs) were used in our in vitro studies. PARP inhibition significantly decreased the markers of oxidative stress and caspase-3 activation and increased smooth muscle actin within plaques from ApoE(-/-) mice fed a high-fat diet. PARP inhibition protected against apoptosis and/or necrosis in SMCs and endothelial cells in response to H(2)O(2) or tumour necrosis factor (TNF). Remarkably, PARP inhibition in FCs resulted in significant sensitization to 7-ketocholesterol (7-KC) by increasing cellular-toxic-free cholesterol, potentially through a down-regulation of acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) expression. 7-KC induced necrosis exclusively in endothelial cells, which was, surprisingly, unaffected by PARP inhibition indicating that PARP inhibition does not prevent all forms of necrotic cell death. In SMCs, PARP-1 inhibition by gene deletion conferred protection against 7-KC or TNF, potentially by reducing caspase-3-like activation, preventing induction of c-Jun N-terminal protein kinase phosphorylation, and inducing extracellular signal-regulated kinase phosphorylation independently of PARP classical enzymatic activity. These data present PARP-1 as an important player in the death of cells constituting atherosclerotic plaques contributing to plaque dynamics. PARP inhibition may be a protective, a neutral, or a sensitizing factor. Additionally, PARP-1 may be a novel factor that can alter lipid metabolism. These novel functions of PARP not only challenge the current understanding of the role of the enzyme in cell death but also provide insights on the intricate contribution of PARP in cellular responses to predominant inflammatory factors within atherosclerotic plaques, presenting additional evidence for the viability of PARP inhibition as a therapeutic strategy for atherosclerosis.

Effectiveness of Increasing Temperature of Rearing Water and Non-feeding against Viral Endothelial Cell Necrosis of Eel

Viral endothelial cell necrosis of eel (VECNE) of Japanese eel Anguilla japonica, caused by an adenovirus (JEAdV), has been a serious problem of aquaculture industry in Japan. In the present study, effectiveness of increasing water temperature and non-feeding against VECNE was evaluated. Cumulative mortalities of fish intraperitoneally injected with 105.05 TCID50/fish of JEAdV increased with elevating water temperature in the range between 20°C and 31°C, but mortality at 35°C was as low as that at 20°C. Rearing infected fish under the non-feeding condition further enhanced the effect of treatment at 35°C. The effectiveness was dependent on rearing periods at the high temperature; more than 3 days at 35°C were needed to reduce mortality. Fish, which survived the primary challenge with JEAdV at 35°C, showed high resistance to re-challenge with JEAdV. From these results, the treatment of fish under the non-feeding condition at 35°C is useful to control VECNE.

Terbinafine‐induced dermatomyositis: a case report and literature review of drug‐induced dermatomyositis

Dermatomyositis, a connective tissue disease syndrome where antibodies to the endothelium of the microvasculature of the skin, muscle and lung are implicated in lesional propagation, is characterized by photodistributed erythema, heliotrope rash, Gottron's papules, muscle weakness and interstitial pulmonary fibrosis. Endotheliotropic viruses and underlying neoplasia are among the inciting triggers. Uncommon drugs, namely the lipid-lowering agents, have been implicated in dermatomyositis. The patient, a 57-year-old man, developed a photodistributed rash and muscle weakness following treatment with the antifungal medication, terbinafine. A skin biopsy was performed, showing an atrophying interface dermatitis with pandermal mucinosis and striking vasculopathic changes including endothelial cell necrosis with denudement and basement membrane zone reduplication. Ultrastructural studies confirmed the presence of endothelial cell injury. Direct immunofluorescent testing showed prominent staining of C5b-9 along the dermal-epidermal junction and within the vasculature. Western blot studies showed strong seroreactivity of his serum to an endothelial-based protein weighing 45,000, a common target described in other microvascular injury-based syndromes. We have shown a temporal association between use of terbinafine and the development of dermatomyositis. The exact basis remains speculative. One potential hypothesis is based on the fact that terbinafine, the active agent in terbinafine, triggers apoptosis of human endothelial cells in culture. Enhanced endothelial cell apoptosis results in the displacement of various cellular antigens creating a state of neoantigenicity; its attendant sequelae is held to be one of anti-endothelial cell antibody formation, a defining pathogenetic event in the evolution of dermatomyositis. The second may be because of the effects of the drug on the promotion of an interferon-rich T-helper-1-dominant cytokine milieu.

Methylglyoxal and high glucose co‐treatment induces apoptosis or necrosis in human umbilical vein endothelial cells

Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). We previously showed that high glucose (HG; 30 mM) or MG (50-400 microM) could induce apoptosis in mammalian cells, but these doses are higher than the physiological concentrations of glucose and MG in the plasma of DM patients. The physiological concentration of MG and glucose in the normal blood circulation is about 1 microM and 5 mM, respectively. Here, we show that co-treatment with concentrations of MG and glucose comparable to those seen in the blood circulation of DM patients (5 microM and 15-30 mM, respectively) could cause cell apoptosis or necrosis in human umbilical vein endothelial cells (HUVECs) in vitro. HG/MG co-treatment directly increased the reactive oxygen species (ROS) content in HUVECs, leading to increases in intracellular ATP levels, which can control cell death through apoptosis or necrosis. Co-treatment of HUVECs with 5 microM MG and 20 mM glucose significantly increased cytoplasmic free calcium levels, activation of nitric oxide synthase (NOS), caspase-3 and -9, cytochrome c release, and apoptotic cell death. In contrast, these apoptotic biochemical changes were not detected in HUVECs treated with 5 microM MG and 30 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers could inhibit 5 microM MG/20 mM glucose-induced cytochrome c release, decrease activation of caspase-9 and caspase-3, and increase the gene expression and protein levels of p53 and p21, which are known to be involved in apoptotic signaling. Inhibition of p53 protein expression using small interfering RNA (siRNA) blocked the activation of p21 and the cell apoptosis induced by 5 microM MG/20 mM glucose. In contrast, inhibition of p21 protein expression by siRNA prevented apoptosis in HUVECs but had no effect on p53 expression. These results collectively suggest that the treatment dosage of MG and glucose could determine the mode of cell death (apoptosis vs. necrosis) in HUVECs, and both ROS and NO played important roles in MG/HG-induced apoptosis of these cells.

Zinc Phthalocyanine Tetrasulfonate (ZnPcS4): A New Photosensitizer for Photodynamic Therapy in Choroidal Neovascularization

The aim of this sutdy was to demonstrate the selective localization of a new photosensitizer, zinc phthalocyanine tetrasulfonate (ZnPcS(4)), in rat eyes and investigate the ability of ZnPcS(4) to produce a photochemical closure of experimental choroidal neovascularization (CNV) upon irradiation with a 670-nm laser light. To determine the biodistribution of ZnPcS(4) and the optimal timing of laser irradiation after photosensitizer administration, fluorescence microscopy with ZnPcS(4) was performed. CNV was created in the fundi of Brown-Norway rats using the argon laser model and documented by fluorescein angiography (FFA) and optical coherence tomography (OCT). Photodynamic therapy (PDT) was performed at the dose of 2.0 mg/m(2) and laser fluences of 600 mW/cm(2) on the CNV and on normal retina and choroid. Treatment outcomes were assessed by FFA and OCT and confirmed by light and electron microscopy. Fluorescence microscopy demonstrated intense ZnPcS(4) fluorescence from the CNV, choriocapillaris, and retinal pigment epithelial cells. Peak ZnPcS(4) intensities in the choriocapillaris and CNV were detected at 10-20 min after an intravenous injection. FFA and OCT indicated that irradiation with 670 nm of laser light 20 min after a ZnPcS(4) injection produced a complete closure of CNV with minimal damage to the overlying retina. Histologic studies, using light and electron microscopy, demonstrated CNV endothelial cell necrosis with minimal damage to the surrounding tissues. ZnPcS(4) selectively localizes to the choriocapillaris and CNV in rats, resulting in the occlusion of laser-induced CNV with minimal damage to the retina tissues. ZnPcS(4) -PDT is a potential new strategy for the treatment of macular degeneration and other human diseases manifesting as CNV.

Panarteritis cutanea benigna – an entity limited to the skin or cutaneous presentation of a systemic necrotizing vasculitis? Report of seven cases and review of the literature

In 1931 Lindberg described a limited and benign subcutaneous form of panarteritis nodosa, which, in contrast to systemic panarteritis, only affects the skin. The terms panarteritis nodosa cutanea benigna, cutaneous polyarteritis nodosa, apoplexia cutanea Freund as well as livedo with nodules are used synonymously for this vasculitis which predominantly affects women in the fifth decade of life. Cutaneous lesions characteristically comprise painful subcutaneous nodules or vasculitis racemosa at the lower extremities. The cutaneous panarteritis may be regarded as its own entity or an isolated skin manifestation within systemic panarteritis nodosa. Seven patients (M=3, F=4) presented with subcutaneous, painful nodules (n=2), erythema and edema (n=2) or livedo racemosa (n=3) on the lower extremities. Histological examination revealed perivascular lymphocyte inflammatory infiltrates with fibrinoid necrosis and bulging of endothelial cells. In a direct immunofluorescence examination neither immunoglobulin nor complement deposits could be found. Serological autoimmune parameters, abdominal ultrasound examination, and chest X-rays showed no systemic manifestation. Erythrocyte sedimentation rate was slightly raised, and hepatitis B/C serology was negative. Topical corticosteroids under occlusion (n=3), oral methyl-prednisolone (n=4) in combination with either azathioprine (n=4) or mycophenolate mofetil (n=2) showed no relapses [follow up 28.43 (6-82) months]. The etiology of panarteritis nodosa cutanea is unknown in detail. Both focal infections and hypersensitivity reactions are discussed. Differential diagnosis includes systemic panarteritis, livedo reticularis, Sneddon's syndrome or erythema nodosum. Despite the prognostically favorable but prolonged chronic course of the disease over decades, patients should be regularly examined to exclude possible transition to systemic disease.

In Vivo Results of a New Focal Tissue Ablation Technique: Irreversible Electroporation

This paper reports results of in vivo experiments that confirm the feasibility of a new minimally invasive method for tissue ablation, irreversible electroporation (IRE). Electroporation is the generation of a destabilizing electric potential across biological membranes that causes the formation of nanoscale defects in the lipid bilayer. In IRE, these defects are permanent and lead to cell death. This paper builds on our earlier theoretical work and demonstrates that IRE can become an effective method for nonthermal tissue ablation requiring no drugs. To test the capability of IRE pulses to ablate tissue in a controlled fashion, we subjected the livers of male Sprague-Dawley rats to a single 20-ms-long square pulse of 1000 V/cm, which calculations had predicted would cause nonthermal IRE. Three hours after the pulse, treated areas in perfusion-fixed livers exhibited microvascular occlusion, endothelial cell necrosis, and diapedeses, resulting in ischemic damage to parenchyma and massive pooling of erythrocytes in sinusoids. However, large blood vessel architecture was preserved. Hepatocytes displayed blurred cell borders, pale eosinophilic cytoplasm, variable pyknosis and vacuolar degeneration. Mathematical analysis indicates that this damage was primarily nonthermal in nature and that sharp borders between affected and unaffected regions corresponded to electric fields of 300-500 V/cm.

Cutaneous manifestations of vasculitis in the dog

Vasculitis is inflammation of a blood vessel wall. It may be a primary event or develop secondary to inflammation, infection, neoplasia and drugs, particularly vaccinations. Vascular conditions involving the skin are uncommon to rare and present the clinician with a diagnostic and therapeutic challenge. In human medicine considerable debate has been given to establishing a consensus about the definition of systemic vasculitides, particularly primary systemic vasculitis syndromes. The classification of such syndromes is based on the size and location of vessels, and the results of immunohistochemical studies implicate a variety of immune mechanisms including allergic (IgE type 1 hypersensitivity), antibody-mediated including antineutrophil cytoplasmic antibodies, immune complex formation and granulomas. In veterinary medicine there are few primary systemic vasculitides recognised. An example is a multisystemic necrotising vasculitis of small vessels in Beagles called juvenile polyarteritis syndrome (JPS). Cutaneous vasculopathies are usually secondary to an underlying process in which (usually) the small vessels - especially the post-capillary venules - are the target of the disease process. Possible secondary causes of cutaneous vascular disease are listed in Table 1. Histologically there is accumulation of cells (macrophages, neutrophils, lymphocytes and plasma cells) within the vessel wall, injury with necrosis and degeneration of endothelial and smooth muscle cells, and fibrin deposition. Eosinophils may be associated with collagen and necrosis of smooth muscle. Vascular diseases involving skin have been subdivided according to the predominant histological pattern. Vasculitides may be cell poor or granulomatous (macrophage is predominant inflammatory cell). Neutrophilic forms may be leukocytoclastic where neutrophil nuclei undergo pyknosis and karyorrhexis - fragmentation, leading

Ultrastructure of skeletal muscle capillaries under conditions of space mission

Capillaries of the rat forepaw skeletal muscles were examined on day 14 of space mission and on days 1 and 14 after landing. Ultrastructural studies revealed apoptosis caused by muscle fiber atrophy and necrobiotic changes eventuating in coagulation or monocellular necrosis of endothelial cells. Formation of capillaries was detected, which can be regarded as an adaptive reaction to injuries caused by space mission factors.

Caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis

The role of caspases and calpain in cisplatin-induced endothelial cell death is unknown. Thus we investigated whether caspases and calpain are mediators of cisplatin-induced apoptosis and necrosis in endothelial cells. Cultured pancreatic microvascular endothelial (MS1) cells were exposed to 10 and 50 microM cisplatin. Apoptosis or necrosis was determined by Hoechst 33342 and propidium iodide (PI) nuclear staining. Cells treated with 10 microM cisplatin had normal ATP levels, increased caspase-3-like activity, excluded PI and demonstrated morphological characteristics of apoptosis at 24 h. Cells treated with 50 microM cisplatin had severe ATP depletion, increased caspase-3-like activity, and displayed extensive PI staining indicative of necrosis at 24 h. There was a dose-dependent increase in caspase-2-like activity and Smac/DIABLO protein. Calpain activity increased significantly with 50 microM, but not 10 microM cisplatin at 24 h. With 50 microM cisplatin, ATP levels were significantly reduced starting at 18 h, caspase-2- and caspase-3-like activities were significantly increased starting at 18 h, and LDH release started at 8 h with maximum increase at 18-24 h. Calpain activity was not increased before 24 h. The increase in LDH release and the nuclear PI staining with 50 microM cisplatin at 24 h was reduced by either the pancaspase inhibitor, Q-VD-OPH, or the calpain inhibitor, PD-150606. Calpain inhibitor had no effect on caspase-3-like activity. In conclusion, in cisplatin-treated endothelial cells, caspases, the major mediators of apoptosis, can also cause necrosis. A calpain inhibitor protects against necrosis without affecting caspase-3-like activity suggesting that calpain-mediated necrosis is independent of caspase-3.

Idiopathic Pulmonary Fibrosis Related to Endothelial Injury and Antiendothelial Cell Antibodies

Mechanisms underlying idiopathic pulmonary fibrosis are not well understood. This paper presents data supporting the hypothesis that microvascular endothelial cell injury and antiendothelial cell antibodies play roles in human idiopathic pulmonary fibrosis. Serologic and pathologic features of 40 patients diagnosed with idiopathic pulmonary fibrosis were evaluated. All patients had open lung biopsies indicating either usual or nonspecific interstitial pneumonitis. All biopsies had morphologic evidence of microvascular injury to the endothelium, and direct immunofluorescence testing revealed variable deposition of IgG, IgM, or IgA within septal microvasculature suggestive of humorally mediated microvascular injury. Ultrastructural studies revealed changes of endothelial cell injury and necrosis and evidence of repetitive episodes of microvascular injury characterized by basement membrane zone collagen deposition and lamellation. Serum samples demonstrated reactivity to multiple endothelial cell antigenic epitopes, and indirect immunofluorescent testing demonstrated a prominent pattern of fluorescence in pulmonary endothelial cell preparations. Serum samples were positive in 37/40 patients for antiphospholipid antibodies with one fourth having positive lupus anticoagulant tests accompanied by thrombotic episodes. In patients with idiopathic pulmonary fibrosis, Factor VIII levels and C-reactive protein levels were also elevated, supporting the presence of endothelial cell injury and inflammation. These data underscore a potential role for immune-based microvascular injury in the evolution of usual or nonspecific interstitial pneumonitis and indicate that those patients have evidence of microvascular injury and endothelial cell necrosis. The high prevalence of antiphospholipid antibodies in these patients may lead to an inherent thrombophilic tendency.

The South African form of severe and complicated canine babesiosis: Clinical advances 1994–2004

Babesia ( canis) rossi infection is common in dogs in South Africa, and frequently causes severe, life-threatening disease. Acidemia, persistent hyperlactatemia, hemoconcentration, elevated creatinine, cerebral babesiosis, pulmonary edema and pancreatitis are all associated with mortality rates above 30%, compared with overall mortality of 12% in admitted cases. Although half the admitted cases are severely anemic, hemoconcentration is associated with far higher mortality. Cerebral babesiosis is uncommon, but carries a poor prognosis. The pathological mechanism has been suggested to be endothelial cell damage and necrosis, followed by segmental microvascular necrosis with perivascular edema and hemorrhage. Renal involvement in babesiosis resembles the functional renal failure of sepsis. Hypotension is common, and other cardiovascular disturbances have been documented. Cerebellar ataxia, rhabdomyolysis and pancreatitis are recently identified complications. While the previous categorization into “severe” (life-threatening anemia) and “complicated” (complications not directly attributable to anemia) disease has proved useful, the distinction is artificial and probably unnecessary. An updated approach to classification is suggested, aimed at grouping animals by severity and prognosis, and using simple measures, such as clinical collapse and abnormal breathing, as much as possible. Although inflammatory mechanisms are undoubtedly important in the pathophysiology of babesiosis, there can be little doubt that tissue hypoxia plays a major role in the disease process.

Effect of Selenium Compounds on the Damage Induced by Oxysterol on Rat Arterial Walls

Wistar rats were fed Se-deficient (0.017 +/- 0.002 mg Se/kg) and Seadequate (0.32 +/- 0.045 Se mg/kg) diets for 12 mo and then were given 5 mg/kg of cholestane-3beta,5alpha,6beta-triol (3-triol), intravenously. Se compounds (Na(2)SeO(3) and ebselen) were supplemented in different doses and times to the Se-deficient rats. Twenty-four hours after 3-triol infusion, the changes in ultrastructures of rat aorta were examined by scanning electron micrography (SEM) and transmission electron micrography (TEM). SEM examinations showed that 3-triol induced diffused injuries on arterial endothelial urfaces of long-term Se-deficient rat, and a large number of holes or craterlike defects were observed. TEM examinations further showed that 3-triol induced swelling, necrosis, and shedding of endothelial cells, which resulted in the destruction of endothelial integrity. Meanwhile, smooth muscle cells proliferated and migrated toward intimae; the breakage of internal elastic lamina benefited the migration of smooth muscle cells. Supplemented with Na(2)SeO(3) (40 microg/kg, 10 d per continuum) and ebselen (20 mg/kg), respectively, exhibited significant protection from damages induced by 3-triol. It seems that protecting mechanisms were different between Na(2)SeO(3) and ebselen. The present investigation gave visual evidence that both injuries induced by cholesterol oxides and the Se nutritional status contributed to the development of atherosclerosis.

Vitamin E Is Essential for Mouse Placentation but Not for Embryonic Development Itself

Vitamin E (alpha-tocopherol) was discovered 80 years ago to be an indispensable nutrient for reproduction in the female. However, it has not been clarified when or where vitamin E is required during pregnancy. We examined the role of alpha-tocopherol in pregnancy using alpha-tocopherol transfer protein (Ttpa)-deficient mice fed specific alpha-tocopherol diets that led to daily, measurable change in plasma alpha-tocopherol levels from nearly normal to almost undetectable levels. A dietary supplement of alpha-tocopherol to pregnant Ttpa-/- (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial development and maturation of the placenta. In addition, exposure to a low alpha-tocopherol environment after initiation of placental formation might result in necrosis of placental syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa(-/-)-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa-/- fetuses were below the detection limit but the fetuses grew normally. These results indicate that alpha-tocopherol is indispensable for the proliferation and/or function of the placenta but not necessary for development of the embryo itself.

Ultrastructure as a Diagnostic Adjunct in the Evaluation of Lung Allograft Biopsies

Humoral immunity contributes significantly to lung graft dysfunction. Recognizing a role of ultrastructural studies in the evaluation and diagnosis of chronic humoral allograft rejection in the kidney, the authors sought to explore its utility as a diagnostic adjunct in lung allograft biopsies. Ultrastructural studies were conducted on 44 biopsies from 26 lung transplant recipients. Endothelial cell activation and necrosis were seen in the setting of acute humoral allograft rejection. Septal chronic vasculopathic changes of thickening and lamellation of the basement membrane zone (BMZ) and BMZ collagen deposition were correlated with greater numbers of humoral allograft rejection episodes and with the development of chronic graft dysfunction/bronchiolitis obliterans syndrome. There was a positive correlation between the extent of septal fibrosis and certain chronic vasculopathic changes, namely collagen deposition in the BMZ and BMZ wrinkling. Patients with chronic graft dysfunction and multiple rejection episodes manifested low diffusion capacities (less than 50% predicted). The results indicate that ultrastructural analysis is useful in identification of septal fibrosis and chronic vasculopathy of the septal microvasculature, correlating with chronic graft dysfunction, encompassing not only fibrotic sequelae of the bronchial wall but also irreversible terminal lung parenchymal changes, the latter associated with repeated episodes of humoral rejection.

Lamisil Induced Dermatomyositis

Background: Dermatomyositis, a connective tissue disease syndrome where antibodies to the endothelium of the microvasculature of the skin, muscle and lung are implicated in lesional propagation, is characterized by photodistributed erythema, heliotrope rash, Gottron’s papules, muscle weakness and interstitial pulmonary fibrosis. Endotheliotropic viruses and underlying neoplasia are among the inciting triggers. Uncommonly drugs, namely the lipid lowering agents, have been implicated in dermatomyositis. Case Report: The patient, a 57‐year‐old male, developed a photodistributed rash and muscle weakness following treatment with the antifungal medication Lamisil. A skin biopsy was performed, showing an atrophying interface dermatitis with pandermal mucinosis and striking vasculopathic changes including endothelial cell necrosis with denudement and basement membrane zone reduplication. Direct immunofluorescent testing showed prominent staining of C5b‐9 along the dermal‐ epidermal junction and within the vasculature. Conclusion: We have shown a temporal association between use of Lamisil and the development of dermatomyositis. Terbinafine, the active agent in Lamisil, promotes apoptosis of human endothelial cells in culture. Enhanced endothelial cell apoptosis results in the displacement of various cellular antigens creating a state of neo‐antigenicity; its attendant sequelae has held to be one of antiendothelial cell antibody formation, a defining pathogenetic event in the evolution of dermatomyositis.

Effect of vessel diameter on the creation of ovine lung radiofrequency lesions in vivo: Preliminary results

We sought to evaluate the effect of radiofrequency ablation (RFA) on pulmonary vessels with respect to potential of injury of these structures, to assess perfusion-mediated "heat sink" effect, and to consider acute and chronic complications. RFAs targeted to perihilar, middle third, and peripheral lung regions were created in vivo in the lung of 10 crossbred sheep. The RITA generator and the Starburst XLi electrode with deployable hooks were used. The approach was open, performed under general anesthesia. Lesions 4 cm in diameter at a target temperature of 80 degrees C were created. Acute (immediate postinterventional euthanasia), subacute (96 h), and chronic (28 days) lesions were evaluated macroscopically, and histologic analysis of the vessels was performed. Patency of the vessels, both arteries and veins, was macroscopically assessed by presence or absence of thrombus and the degree of vascular injury and the viability of perivascular pneumocytes as well as endobronchial injury were histologically assessed. In the acute, subacute, and chronic setting, heat sink effect, indicated by invagination of the tissue between vessel and ablated region, was only observed in vessels greater than 3 mm in diameter. Thrombus was seen in 20% of the vessels smaller than 3 mm. On histopathology, vessels smaller than 3 mm showed at least partial vessel wall injury, characterized by endothelial cell necrosis and luminal thrombus. In the vessels greater than 3 mm the extent of vessel wall injury decreased with increasing vessel diameter. No acute complications were noted. For the chronic complications a bronchopleural fistula and a lung abscess were found. There seems to be a narrow transition zone for pulmonary vessels around 3 mm, beyond which the heat sink effect was seen consistently and substantial vascular injury was rare.

Synergistic Antitumor Activity of Histamine Plus Melphalan in Isolated Limb Perfusion: Preclinical Studies

We have previously shown how tumor response of isolated limb perfusion (ILP) with melphalan was improved when tumor necrosis factor alpha (TNF-alpha) was added. Taking into account that other vasoactive drugs could also improve tumor response to ILP, we evaluated histamine (Hi) as an alternative to TNF-alpha. We used a rat ILP model to assess the combined effects of Hi and melphalan (n = 6) on tumor regression, melphalan uptake (n = 6), and tissue histology (n = 2) compared with Hi or melphalan alone. We also evaluated the growth of BN-175 tumor cells as well as apoptosis, necrosis, cell morphology, and paracellular permeability of human umbilical vein endothelial cells (HUVECs) after Hi treatment alone and in combination with melphalan. The antitumor effect of the combination of Hi and melphalan in vivo was synergistic, and Hi-dependent reduction in tumor volume was blocked by H1 and H2 receptor inhibitors. Tumor regression was observed in 66% of the animals treated with Hi and melphalan, compared with 17% after treatment with Hi or melphalan alone. Tumor melphalan uptake increased and vascular integrity in the surrounding tissue was reduced after ILP treatment with Hi and melphalan compared with melphalan alone. In vitro results paralleled in vivo results. BN-175 tumor cells were more sensitive to the cytotoxicity of combined treatment than HUVECs, and Hi treatment increased the permeability of HUVECs. Hi in combination with melphalan in ILP improved response to that of melphalan alone through direct and indirect mechanisms. These results warrant further evaluation in the clinical ILP setting and, importantly, in organ perfusion.

THE HEPATIC EXTRACELLULAR MATRIX DURING COLD ISCHEMIA/WARM REPERFUSION OF THE RAT LIVER: ROLE OF MATRIX METALLOPROTEINASES IN ITS ALTERATION.

A310 Background: Poor initial graft function remains a major clinical problem in human liver transplantation. Its incidence (15 to 25% of transplanted patients) appears to depend on the length of cold storage and has thus been ascribed to injury from harvesting, cold storage and warm reperfusion after blood vessel reconnection; i.e. cold ischemia/warm re-perfusion (CI-WR) injury. The underlying mechanism of CI-WR injury has been studied extensively over the last decade, but it remains poorly understood. Recently, it has been demonstrated that different time courses and mechanisms of cell death occur in rat livers after CI-WR, with early sinusoidal endothelial cell necrosis followed by delayed hepatocyte apoptosis. These processes did not appear to be of major importance in the mechanism of graft failure since both were similar under non-lethal and lethal conditions. By contrast, prolonged CI was associated with an alteration of the extracellular matrix (ECM), leading to extensive coagulative necrosis of hepatocytes as found in conditions ending in graft failure. Aims: Since it is known that warm ischemia/WR of livers induces an increased local synthesis of metalloproteinases (MMPs), proteases implicated in the degradation of the hepatic ECM, we looked if the same phenomenon was occurring during prolonged CI/WR of rat livers ensuing in ECM disappearance responsible for the graft malfunction. Methods: Rat livers were preserved for 0, 24 (viable livers) and 42 h (non-viable livers) in the UW solution (4°C), then reperfused for 0, 10, 20, 30, 40, 60, 90, and 120 minutes with a krebs buffer solution (37°C) containing 20% erythrocytes. The first 15 mLs of the liver rinsing solution were taken during early WR and frozen liver biopsies were obtained at the end of each period (n=4 in all groups). Total liver RNAs and proteins were extracted, then MMP and their natural inhibitor (TIMP) expression was analysed by Northern Blot quantitative analysis and MMP activity was nalyzed using a fluorescent substrate (MCA-MAT) or by zymography. Results: although most of these genes are not expressed in the normal rat liver, there was an induction of MMP-13 and 14 expression as well as of TIMP-1 and 2 expression in all livers, independently from CI or WR periods. There was also an increased MMP activity in all livers, as measured ding a in presence or absence of an inhibitor (BB94), whatever the experimental condition. An increased activity of MMPs (particularly gelatinases A and B) was found in the initial flush solution. Comments: These data are quite different from those observed following warm ischemia/WR, particularly when considering that, following CI, the ECM rapidly deteriorates with WR. However since this phenomenon occurs very early during WR, it might be due to the liberation of MMPs already present in sinusoidal endothelial cells and released by their necrosis during prolonged CI, as suggested by our data and others. Conclusion: Induction of hepatic MMP synthesis does not occurs following prolonged CI/WR of rat livers and thus has no major role in the alteration of the hepatic extracellular matrix observed under these conditions. This phenomenon might result from the local passive release of preformed MMPs from dead hepatic cells.