Vascular endothelial growth factor overexpression does not enhance adipose stromal cell-induced protection on muscle damage in critical limb ischemia.

OBJECTIVES: Critical limb ischemia complicates peripheral artery disease leading to tissue damage and amputation. We hypothesized that modifying adipose stromal cells (ASCs) to overexpress human vascular endothelial growth factor 165 (VEGF) would limit ischemic muscle damage to a larger extent than nonmodified ASCs. APPROACH AND RESULTS: Rabbits with critical hindlimb ischemia were injected with allogeneic abdominal fat-derived ASCs transfected with plasmid-VEGF165 (ASCs-VEGF; n=10). Additional rabbits received nontransfected ASCs (ASCs; n=10) or vehicle (placebo; n=10). One month later, ASCs-VEGF rabbits exhibited significantly higher density of angiographically visible collaterals and capillaries versus placebo (both P<0.05) but not versus ASCs (both P=NS). Arteriolar density, however, was increased in both ASCs and ASCs-VEGF groups (both P<0.05 versus placebo). ASCs-VEGF and ASCs showed comparable post-treatment improvements in Doppler-assessed peak systolic velocity, blood pressure ratio, and resistance index. Ischemic lesions were found in 40% of the muscle samples in the placebo group, 19% in the ASCs-VEGF group, and 17% in the ASCs groups (both P<0.05 versus placebo, Fisher test). CONCLUSIONS: In a rabbit model of critical limb ischemia, intramuscular injection of ASCs genetically modified to overexpress VEGF increase angiographically visible collaterals and capillary density. However, both modified and nonmodified ASCs increase arteriolar density to a similar extent and afford equal protection against ischemia-induced muscle lesions. These results indicate that modifying ASCs to overexpress VEGF does not enhance the protective effect of ASCs, and that arteriolar proliferation plays a pivotal role in limiting the irreversible tissue damage of critical limb ischemia.

Severe mitral regurgitation after radiotherapy.

We present the case of a 69-year-old patient with a history of gynecological neoplasia and a pulmonary metastasis, who in 1996 underwent chemotherapy and mediastinal radiotherapy followed by cancer remission. Ten years later she presented with heart failure and her Doppler echocardiogram showed severe mitral regurgitation with pulmonary hypertension. In 2011, she underwent a mitral valve replacement with a biological prosthesis and the pathology exam revealed valve damage consistent with radiotherapy-induced changes. This unusual mechanism of mitral regurgitation can be demonstrated clearly by echocardiography and should be disseminated among cardiology physicians and in patients who have survived for long periods after radiotherapy, it is important to remember that cardiac complications may indeed occur, and the treating physician is responsible for detecting them.

Effect of vascular endothelial growth factor gene transfer on infarct size, left ventricular function and myocardial perfusion in sheep after 2 months of coronary artery occlusion.

BACKGROUND: In large mammalian models of acute myocardial infarction (AMI), plasmid-mediated vascular endothelial growth factor (pVEGF) gene transfer has been shown to induce angio-arteriogenesis, proliferation of myocyte precursors and adult cardiomyocyte mitosis, reducing infarct size at 15 days after coronary artery occlusion. However, it is unknown whether these effects persist at longer follow-up times, nor how they affect cardiac performance. We thus assessed infarct size, left ventricular (LV) function and perfusion in 2-month-old ovine AMI. METHODS: Adult sheep with coronary artery occlusion were randomized to blindly receive ten intramyocardial injections of 3.8 mg of pVEGF or empty plasmid distributed at the infarct border. Three and 60 days later, LV perfusion (single-photon emission computed tomography) and function (stress echocardiography) were assessed. Finally, hemodynamics (LV catheterization), scar size and peri-infarct histology were studied. RESULTS: Infarct size was 30% smaller in pVEGF-treated sheep (23.6 ± 1.9% versus 32.7 ± 2.7% of the LV; p < 0.02). Percentage fractional shortening and wall thickening at the infarct border improved after pVEGF, as did myocardial perfusion and LV wall motion under pharmacological stress. Global LV function did not differ between groups, although the force-frequency response was preserved in pVEGF group and lost in placebo animals. These effects were associated with angio-arteriogenesis and proliferation of cardiomyocyte precursors. CONCLUSIONS: In sheep with AMI, pVEGF gene transfer affords long-term infarct size reduction, yielding regional LV function and perfusion improvement and reducing remodeling progression. These results suggest the potential usefulness of this approach in the clinical setting.

The heavy chain variable segment gene repertoire in chronic Chagas' heart disease.

Patients chronically infected with Trypanosoma cruzi develop chronic Chagas' heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas' disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.

Coronary microcirculation remodeling in patients with idiopathic dilated cardiomyopathy.

BACKGROUND: In patients with idiopathic dilated cardiomyopathy (IDCM), the myocardial blood flow is markedly depressed. The presence of alterations in the number and length of the coronary microcirculation has not been explored. METHODS: In explanted hearts of 6 patients with IDCM and in 6 normal control hearts, the arteriolar length density and capillary numerical density were determined in sections stained with orcein (vessels 50-200 µm in diameter), for smooth muscle actin (vessels 6-50 µm in diameter) and CD34 (capillaries). RESULTS: No difference with normal hearts was observed in capillary numerical density and in length density of vessels above 50 µm in diameter. In IDCM, more than 50% of arterioles below 50 µm in diameter displayed an incomplete smooth muscle wall, and length density, especially for arterioles between 6 and 20 µm in diameter, was significantly lower (42.84 ± 8.51 mm/mm(3)) than in controls (75.34 ± 3.05 mm/mm(3), p = 0.001). CONCLUSIONS: In IDCM, arterioles below 50 µm in diameter display an incomplete smooth muscle wall and a significant decrease in length density. These observations provide an anatomical basis for a decreased coronary flow reserve in IDCM.

[Accuracy of multislice computed tomography in the diagnosis of hepatocellular carcinoma in patients with cirrhosis evaluated for liver transplantation]. / Precisión de la tomografía multidetector en el diagnóstico de hepatocarcinoma en pacientes con cirrosis evaluados para trasplante hepático.

BACKGROUND: The presence of hepatocellular carcinoma (HCC) within Milan criteria provides additional points in the MELD (Model for end stage liver disease) system and benefits in the order of organ allocation. The imaging methods play a key role in this process and represent an essential tool in the diagnosis and staging of HCC. OBJECTIVES: 1) To assess the accuracy of dynamic multidetector computed tomography (MDCT) in the diagnosis of HCC in patients with cirrhosis who are listed for liver transplantation. 2) To evaluate the diagnostic performance of TCMD in relation to tumor size. MATERIAL AND METHODS: We retrospectively reviewed the reports of MDCT performed in our institution to 62 patients who were then transplanted The histological analysis of explants was considered as the reference method in the diagnosis of HCC. MDCT studies were performed with dynamic protocol in arterial, portal and late phases. RESULTS: Dynamic MDCT showed a sensitivity of 87.5% and correctly characterized 28 of 35 patients with pathology proved hepatocellular carcinoma. MDCT was negative in 25 of 30 patients without hepatocellular carcinoma in the explanted liver, with a specificity of 83.3%. Nodule by nodule evaluation revealed a sensitivity of 80.3% and a specificity of 72.2%. CONCLUSION: In our center MDCT presented high accuracy in the correct diagnosis of HCC, showing its reliability when requesting additional points for organ allocation.

Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation.

BACKGROUND: One hundred years after the discovery of Chagas disease, it remains a major neglected tropical disease. Chronic Chagas heart disease (cChHD) is the most severe manifestation. Heart transplantation is the proper treatment for end-stage heart failure, although reactivation of disease may result after receipt of immunosuppressive therapy. T. cruzi strains cluster into 6 discrete typing units (DTUs; I-VI) associated with different geographical distribution, transmission cycles and varying disease symptoms. In the southern cone of South America, T. cruzi II, V, and VI populations appear to be associated with Chagas disease and T. cruzi I with sylvatic cycles. METHODS: Molecular characterization of DTUs, T. cruzi I genotypes (on the basis of spliced-leader gene polymorphisms), and minicircle signatures was conducted using cardiac explant specimens and blood samples obtained from a cohort of 16 Argentinean patients with cChHD who underwent heart transplantation and from lesion samples obtained from 6 of these patients who presented with clinical reactivation of Chagas disease. RESULTS: Parasite persistence was associated with myocarditis progression, revealing T. cruzi I (genotype Id) in 3 explant samples and T. cruzi II, V, or VI in 5 explant samples. Post-heart transplantation follow-up examination of bloodstream DTUs identified T. cruzi I in 5 patients (genotypes Ia or Id) and T. cruzi II, V, or VI in 7 patients. T. cruzi I, V, and VI were detected in skin chagoma specimens, and T. cruzi V and VI were detected in samples obtained from patients with myocarditis reactivations. Multiple DTUs or genotypes at diverse body sites and polymorphic minicircle signatures at different cardiac regions revealed parasite histotropism. T. cruzi I infections clustered in northern Argentina (latitude, 23 degrees S-27 degrees S), whereas T. cruzi II, V, or VI DTUs were more ubiquitous. CONCLUSIONS: Multiple DTUs coexist in patients with Chagas disease. The frequent finding of T. cruzi I associated with cardiac damage was astounding, revealing its pathogenic role in cChHD at the southern cone.

Long-term effects of growth hormone on infarct size and left ventricular function in sheep with coronary artery occlusion.

The effects of growth hormone (GH) on infarct size and left ventricular (LV) function in experimental acute myocardial infarction (AMI) have been controversial. Moreover, little, if any, information exists regarding long-term evaluation of therapeutic doses of GH in large mammalian models of AMI. We therefore aimed to assess the effect of therapeutic doses of GH over 3.5 months on infarct size and heart function in sheep with AMI. After coronary artery ligation, sheep received subcutaneous human GH 8 IU/d (n = 8) or vehicle (n = 8) over 100 days. Infarct area was similar in GH (16.9% +/- 3% of LV area) and placebo (16.5% +/- 3.7%, P = not significant) sheep. At 3 days of treatment onset, but not at later times, GH sheep had higher LV shortening fraction (30.7% +/- 3.5% vs. 24.8% +/- 6.1%, P < 0.04), systolic anterior wall thickness (10.1 +/- 0.8 vs. 8.6 +/- 1.2 mm, P < 0.02), and cardiac index (3.8 +/- 0.6 vs. 2.8 +/- 0.7 L x min x m, P < 0.01). This evolution of function parameters paralleled that of serum insulin-like growth factor 1 levels, which differed significantly only during the first week, suggesting a direct effect of GH on LV contractility. These results may suggest the usefulness of therapeutic doses of GH at the early phases of AMI but do not support maintaining the treatment for longer time.

Presence of vulnerable coronary plaques in middle-aged individuals who suffered a brain death.

AIMS: Vulnerable plaques in coronary arteries are frequently found in individuals who died suddenly or due to an acute coronary syndrome. The prevalence and characteristics of these plaques in the middle-aged apparently healthy population are unknown. METHODS AND RESULTS: From a total of 652 hearts from transplant donors collected between 1996 and 2007, we selected those from apparently healthy individuals older than 40 years old who died of head trauma or stroke and had no evidence of prior vascular diseases. The coronary arteries were examined by serial sectioning at 3 mm intervals, and all areas of cross-sectional luminal narrowing were processed for histological, immunohistochemical, and morphometric studies. The atherosclerotic plaques were classified according to the American Heart Association Report. A total of 160 hearts were examined. Mean age was 50.3 +/- 5.8 years. Sixty-eight hearts had no advanced coronary atherosclerotic lesions (Type I, II, and III of the American Heart classification). In the remaining 92 hearts, we found 179 plaques considered high-risk lesions (American Heart Association Type IV, V, and VI). These plaques were more frequently found in males (P < 0.001) and in those with a higher heart weight (P < 0.001). The median (25th and 75th percentiles) vascular narrowing value using a planimetric analysis was 32% (21-53). No significant association with the cause of death was found (P = 0.09). CONCLUSION: High-risk coronary artery plaques not associated with significant vascular lumen reduction exist in 57% of patients who suffered a brain death with a mean of 1.11 lesions prone to rupture per individual.

Advances in coronary heart disease surgery in Latin America.

BACKGROUND: The beginnings of coronary artery bypass graft in Latin America could be set in the year 1971. Since then, improvements in technique and greater experience have resulted in a rapid increase in the rate of interventions performed in the region. METHODS AND RESULTS: Searches through PubMed and Literatura Latinoamericana y del Caribe en Ciencias de la Salud, as well as personal communications from specialists from Latin America, have been the source of information. Articles were selected by their content related to the theme, and the authors' nationality and information is mainly from Latin America. Demographic information of the population of Latin America denotes higher age averages, and this implies an increase in the severity of comorbidities in patients who undergo surgery. Longer life expectancy and improvements in medical therapy have implied that patients survive a first intervention beyond the expected time a bypass persists patent. Wall vessel properties of arterial conduits, plus a better anastomotic technique, seem to be the current solution to worsening in the coronary health of patients who undergo revascularization surgery in Latin America. CONCLUSIONS: Despite scarce economic investment in medical sciences, many academic groups contribute to the exploration of therapeutic pharmacological combinations and inclusively apply genetic strategies.

Expression of the MDR-1 gene-encoded P-glycoprotein in cardiomyocytes of conscious sheep undergoing acute myocardial ischemia followed by reperfusion.

We have recently reported that in chronic myocardial ischemia, adult mammalian cardiomyocytes express P-glycoprotein (P-gp). We now investigate if P-gp is also expressed in acute regional ischemia followed by reperfusion. Adult conscious sheep underwent 12-min occlusion of the mid-left anterior descending artery (inflatable cuff). Successful ischemia-reperfusion was confirmed by monitoring percent systolic left ventricular anterior wall thickening (sonomicrometry) during the whole ischemic period and every 10 min over 2 hr following cuff deflation. At 3, 24, and 48 hr after reperfusion, P-gp expression was investigated by immunohistochemistry and Western blot and MDR-1 mRNA by RT-PCR. Cardiomyocytes in the occluded artery territory (but not those in remote areas) consistently expressed P-gp at their sarcolemma. Whereas at 3 and 24 hr P-gp was mainly observed in the T tubules, at 48 hr it predominated in intercalated discs and gap junctions. RT-PCR and Western blot revealed higher expression in ischemic than in control myocardium. We conclude that in adult sheep with acute myocardial ischemia, the MDR-1 gene-encoded P-gp is expressed at the sarcolemma of the cardiomyocytes from 3 hr up to at least 48 hr after reperfusion.

Endomitosis and polyploidization of myocardial cells in the periphery of human acute myocardial infarction.

OBJECTIVE: Although the genetic program for reinitiating DNA synthesis exists in post-mitotic cardiomyocytes, and it was reported that in human acute myocardial infarction (AMI) a significant proportion of myocytes enter mitosis, the rule is that the lost tissue is replaced by a collagen scar. The purpose of this study was to search for the basis of this discordance in order to devise future strategies to induce division of myocytes into daughter cells that may replace the lost tissue with contractile cells. METHODS: In 15 human hearts with 1- to 21-day-old infarcts, the expression of the cell cycle proteins Ki67 antigen, cyclins D, A, and B1, the presence of mitotic bodies, and the ploidy status were investigated with immunoenzymatic methods, light and laser confocal microscopy, and densitometry in the myocytes surrounding the infarct area. RESULTS: In 7- to 13-day-old infarcts, 11.61+/-6.94% of the myocytes presented Ki67+ nuclei, and a lower proportion presented cyclins D, A, and B. At earlier and later times, the proportion of Ki67+ myocytes was significantly lower. Although under confocal microscopy and fluorescent labels, some of the Ki67+ myocytes appeared to be in different stages of mitosis, with Nomarski optics and hematoxylin counterstaining, the condensed chromosomes, although arranged in metaphase and anaphase plates or split in sister chromatids, were always located within a preserved nuclear envelope, indicating the presence of endomitosis. Conventional mitosis was exceptionally observed. In the 14- and 21-day-old infarcts, the ploidy of the myocytes adjacent to the infarct was significantly higher than in distant zones. CONCLUSION: These observations indicate that in human infarcts, entrance of cardiomyocytes into the cell cycle is transient and that endomitosis, leading to polyploidy, rather than mitosis, leading to karyokinesis, is the final fate of cycling cells. Both observations may account for the discordance between the regenerative ability of myocytes and the lack of an efficient reparative process in human AMI.

Activated macrophages as a feeder layer for growth of resident cardiac progenitor cells.

The adult heart contains a population of cardiac progenitor cells (CPCs). Growing and collecting an adequate number of CPCs demands complex culture media containing growth factors. Since activated macrophages secrete many growth factors, we investigated if activated isolated heart cells seeded on a feeder layer of activated peritoneal macrophages (PM) could result in CPCs and if these, in turn, could exert cardioprotection in rats with myocardial infarction (MI). Heart cells of inbred Wistar rats were isolated by collagenase digestion and cultured on PM obtained 72 h after intraperitoneal injection of 12 ml thioglycollate. Cells (1 × 10(6)) exhibiting CPC phenotype (immunohistochemistry) were injected in the periphery of rat MI 10 min after coronary artery occlusion. Control rats received vehicle. Three weeks later, left ventricular (LV) function (echocardiogram) was assessed, animals were euthanized and the hearts removed for histological studies. Five to six days after seeding heart cells on PM, spherical clusters composed of small bright and spherical cells expressing mostly c-Kit and Sca-1 antigens were apparent. After explant, those clusters developed cobblestone-like monolayers that expressed smooth muscle actin and sarcomeric actin and were successfully transferred for more than ten passages. When injected in the MI periphery, many of them survived at 21 days after coronary ligature, improved LV ejection fraction and decreased scar size as compared with control rats. CPC-derived cells with cardiocyte and smooth muscle phenotypes can be successfully grown on a feeder layer of activated syngeneic PM. These cells decreased scar size and improved heart function in rats with MI.

Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia-Inducible Factor 1-α (HIF1-α) in an Ovine Model of Acute Myocardial Infarction.

BACKGROUND: Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-α (HIF1-α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI. METHODS AND RESULTS: Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-α (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P<0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-α-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF. CONCLUSIONS: Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.

Sialic acid as a protective barrier against neointima development.

Arterial sialic acid (SA) has been shown to attenuate the binding of fibrinogen and low-density lipoproteins (LDL) to the vessel wall, presumably protecting against atherosclerosis. This study was aimed to assess the effect of changes in SA content in intimal thickening, an early step in the development of atherosclerosis. New Zealand white rabbits were subjected to bilateral carotid periarterial collaring, followed by in situ-perfusion with neuroaminidase (random artery) and with vehicle (contralateral control artery). The efficiency of SA removal was evaluated in perfusates and arterial homogenates, and arterial tissue samples were obtained 7 and 14 days after the intervention to assess morphological changes. Neuraminidase significantly reduced SA by 16.7%. Arterial desialylation was associated with a significantly increased neointimal formation. Proliferation of smooth muscle cells (SMCs), assessed by incorporation of bromo-2'-deoxyuridine into replicating DNA was also significantly increased in desialylated arteries. In addition, immunohistochemical studies showed a slightly stronger oxidized-LDL (ox-LDL) immunostaining in neointima of desialylated arteries than in control vessels. A mild reduction of SA increases intimal thickening, at least partly due to an enhanced proliferation of SMCs, and may facilitate the accretion of atherogenic lipoproteins, providing evidence for the potential role of SA in the protection against neointimal development.

Cardiomyocytes of chronically ischemic pig hearts express the MDR-1 gene-encoded P-glycoprotein.

The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.

Effects of short-term inhaled nitric oxide on interleukin-8 release after single-lung transplantation in pigs.

BACKGROUND: Lung transplantation has evolved to become an effective treatment for a variety of end-stage lung diseases. However, severe reperfusion injury is still a major cause for postoperative morbidity and mortality. Although lung reperfusion injury is complex and has not been fully comprehended yet, neutrophil infiltration and cytokine activation have been postulated to play a main role. Recent studies showed that nitric oxide (NO) therapy has salutary effects on lung chronic and acute pathologies because it inhibits interleukin-8 (IL-8) release, but no data have been found on its effects during organ harvest. The aim of this study was to assess whether low doses of inhaled NO pre-treatment at the time of harvesting improves allograft function during early reperfusion in a porcine model. METHODS: Twenty-two Landrace pigs were randomly assigned to NO-treated and control groups. In NO-treated pigs, NO at 20 ppm was administered 30 min before harvest. During the early allograft reperfusion period IL-8 content, dynamic and static compliance and gas exchange (Pa/FiO2 and PaO2) were measured in both control and NO-treated lungs. RESULTS: Pre-treatment with NO at the time of harvesting showed improvement of allograft function in terms of dynamic (92 +/- 8% in NO vs 72 +/- 7% in the control group, p < .05) and static (83 +/- 8% in NO vs 63 +/- 7% in the control group, p < 0.05) compliance and gas exchange (PaO2: 96 +/- 4% in NO vs 74 +/- 4.5% in the control group, p < 0.01; Pa/FiO2: 97 +/- 5% in NO vs 74 +/- 5% in the control group, p < 0.01) by diminishing IL-8 (66.5 +/- 4.7 pg/ml in NO versus 208 +/- 43 pg/ml in the control group, p < 0.05) release in pigs. CONCLUSION: These results show for the first time that NO pre-treatment at the time of harvesting reduces allograft reperfusion injury in part due to its effects on IL-8 release.

Arteriogenesis induced by intramyocardial vascular endothelial growth factor 165 gene transfer in chronically ischemic pigs.

Exogenous vascular endothelial growth factor (VEGF) improves tissue perfusion in large animals and humans with chronic myocardial ischemia. Because tissue perfusion is mainly dependent on the arteriolar tree, we hypothesized that the neovascularizing effect of VEGF should include arteriogenesis, an effect not as yet described in large mammalian models of myocardial ischemia. In the present study we investigated the effect of intramyocardial plasmid-mediated human VEGF(165) gene transfer (pVEGF(165)) on the proliferation of vessels with smooth muscle in a pig model of myocardial ischemia. In addition, we assessed the effect of treatment on capillary growth, myocardial perfusion, myocardial function and collateralization. Three weeks after positioning of an Ameroid constrictor (Research Instruments SW, Escondido, CA) in the left circumflex artery, pigs underwent basal perfusion (single-photon emission computed tomography [SPECT] with (99m)Tc-sestamibi) and regional function (echocardiography) studies at rest and under dobutamine stress, and were then randomly assigned to receive transepicardial injection of pVEGF(165) 3.8 mg (n = 8) or placebo (empty plasmid, n = 8). All experimental steps and data analysis were done in a blinded fashion. Five weeks later, pVEGF(165)-treated pigs showed a significantly higher density of small (8-50 microm in diameter) vessels with smooth muscle, higher density of capillaries, and improved myocardial perfusion. These results indicate an arteriogenic effect of VEGF in a large mammalian model of myocardial ischemia and encourage the use of VEGF to promote arteriolar growth in patients with severe coronary artery disease.

Human regional lymph nodes draining cancer exhibit a profound dendritic cell depletion as comparing to those from patients without malignancies.

Dendritic cells (DCs) are bone-marrow derived 'professional' antigen presenting cells (APC). They are considered as the most potent APC able to induce primary immune responses. DC efficiently capture and process proteic and non-proteic antigens. They are widely distributed throughout the body and occupy sentinel positions such as epithelia. Establishment of an immune response against cancer may depend of the capacity of DCs to transfer (to capture, to process and to present) tumor antigens into regional lymph nodes where they can induce a specific response leading to tumor rejection. Because host 'professional' DCs are one of the most important elements in the induction of specific anti-tumor responses and lymph nodes are the places where the immune response takes place, we investigated the densities of DCs within regional metastasis-free lymph nodes from 47 patients with different malignant epithelial tumors as comparing with lymph nodes from 11 patients without malignancies using an immunohistochemistry method with anti-S100 protein, CD86 and CD1a antibodies. By means of morphometric analysis, we observed that S100+ and CD1a+ DCs densities in regional lymph nodes from cancer patients were significatively decreased as compared with control lymph nodes (P<0.0001 and 0.003, respectively). S100+ DCs and CD86+ DCs densities in lymph nodes draining cancer were similar. Taken together, these data indicated that lymph nodes draining cancer had significantly less CD1a+ DCs than S100+ and possibly CD86+ DCs. These findings may represent another mechanism by which tumors evade the immune recognition.

DNA content and expression of cell cycle proteins in caterpillar nuclei from fetal human cardiac myocytes.

Caterpillar nuclei (CN) are characterized by their peculiar morphology, with chromatin distributed in clusters and running along the longitudinal axis of the nucleus. They can be observed in normal hearts of fetuses as well as in hearts of children and adults with rheumatic heart disease. This study has demonstrated by means of ploidy studies with digital image analysis that in the fetal heart (20.5+/-1.8 weeks) the CN (diploid = 5.6+/-8.4%; tetraploid = 46.2+/-24.2%; hypertetraploid = 46.9+/-26.3%) present higher DNA content than non-caterpillar myocyte nuclei (diploid = 89.4+/-6.2%; tetraploid = 10.0+/-4.1%; hypertetraploid = 1.5+/-1.3%) (P=0.000001, 0.00013, and 0.000038, respectively). Expression of proliferation cell nuclear antigen (PCNA; 30.6+/-11.7% in CN and 13.4+/-7.3% in non-caterpillar myocyte nuclei; P=0.0115) and cyclin B1 (2.8+/-3.8% and 12.6+/-15.6%, respectively; P=n.s.) was also positive in these nuclei. In conclusion, these results suggest that there exists a relationship between CN morphology and myocyte replication phenomena.