Inactivity of nitric oxide synthase gene in the atherosclerotic human carotid artery.

OBJECTIVE: Nitric oxide (NO) inhibits thrombus formation, vascular contraction, and smooth muscle cell proliferation. We investigated whether NO release is enhanced after endothelial NO synthase (eNOS) gene transfer in atherosclerotic human carotid artery ex vivo. METHODS AND RESULTS: Western blotting and immunohistochemistry revealed that transduction enhanced eNOS expression; however, neither nitrite production nor NO release measured by porphyrinic microsensor was altered. In contrast, transduction enhanced NO production in non-atherosclerotic rat aorta and human internal mammary artery. In transduced carotid artery, calcium-dependent eNOS activity was minimal and did not differ from control conditions. Vascular tetrahydrobiopterin concentrations did not differ between the experimental groups. Treatment of transduced carotid artery with FAD, FMN, NADPH, L-arginine, and either sepiapterin or tetrahydrobiopterin did not alter NO release. Superoxide formation was similar in transduced carotid artery and control. Treatment of transduced carotid artery with superoxide dismutase (SOD), PEG-SOD, PEG-catalase did not affect NO release. CONCLUSIONS: eNOS transduction in atherosclerotic human carotid artery results in high expression without any measurable activity of the recombinant protein. The defect in the atherosclerotic vessels is neither caused by cofactor deficiency nor enhanced NO breakdown. Since angioplasty is performed in atherosclerotic arteries,eNOS gene therapy is unlikely to provide clinical benefit.

Sterol regulatory element binding proteins downregulate LDL receptor-related protein (LRP1) expression and LRP1-mediated aggregated LDL uptake by human macrophages.

OBJECTIVE: In the extracellular intima, extracellular matrix proteoglycans favor LDL retention and aggregation (agLDL). In contrast to native LDL (nLDL), agLDL induces high intracellular cholesteryl ester (CE) accumulation in macrophages. It has been suggested that LDL receptor-related protein (LRP1) is involved in agLDL binding and internalization by macrophages. The aim of this work was to analyze whether sterol regulatory element binding proteins (SREBPs) modulate LRP1 expression and LRP1-mediated agLDL uptake by human monocyte-derived macrophages (HMDM). METHODS AND RESULTS: The treatment of HMDM with small anti-LRP1 interfering RNA (siRNA-LRP1) led to the specific inhibition of LRP1 mRNA expression and also to the inhibition of LRP1 protein expression in these cells. In siRNA-LRP1-treated HMDM, CE accumulation from agLDL uptake (84.66+/-5 microg CE/mg protein) was reduced by 95.76+/-5.22%. This suggests that LRP1 plays a pivotal role in agLDL uptake by HMDM. N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of SREBP catabolism, maintained high levels of active SREBP-2 and SREBP-1 even in the presence of nLDL and agLDL. Therefore, ALLN induced LDL receptor (LDLR) upregulation. Concomitantly, a strong downregulation of LRP1 mRNA and LRP1 protein was observed in ALLN-treated macrophages. By decreasing LRP1 expression levels, ALLN reduced CE accumulation from agLDL at all tested concentrations. CONCLUSIONS: These results suggest that high levels of active SREBPs downregulate LRP1 expression and intracellular CE accumulation in HMDM.

[Neovascularization in human coronary arteries with lesions of different severity]. / Neovascularización en arterias coronarias humanas con distintos grados de lesión.

INTRODUCTION AND OBJECTIVES: Endothelial function can be modulated by growth factors produced by activated smooth muscle cells, inflammatory cells and plasma products that infiltrate the lesion. The aim of this study was to quantify neovessels in human coronary arteries with atherosclerotic lesions of different severity and analyze their relationship with inflammatory cell and plasma product infiltrates. PATIENTS AND METHOD: We studied 60 coronary arteries from patients who underwent heart transplant. Cellular markers (smooth muscle cell, monocyte/macrophage), the presence thrombin/prothrombin and expression of vascular endothelial growth factor (VEGF) were analyzed and quantified by conventional histology, immunohistochemistry and image analysis techniques. RESULTS: Neovessels were detected in advanced lesions, and a positive correlation was observed with the degree of vessel remodeling, monocyte/macrophage infiltration and lipid deposition. Smooth muscle cells were the main producers of VEGF in both the intima and media layers of advanced lesions. In these lesions thrombin/prothrombin-positive areas colocalized with activated smooth muscle cells. CONCLUSIONS: The presence of neovessels in coronary arteries correlated with inflammatory cell infiltration, lipid deposition and thrombin/prothrombin content. VEGF expression was mainly associated with smooth muscle cells, indicating a key role of these cells in the modulation of endothelial cell function.

Neovascularización en arterias coronarias humanas con distintos grados de lesión / Neovascularization in human coronary arteries with lesions of different severity

Introducción y objetivos. Los factores de crecimiento producidos por las células musculares lisas activadas, las células inflamatorias y especies moleculares plasmáticas infiltradas en la lesión pueden modular la función endotelial. El objetivo de este estudio ha sido cuantificar la presencia de neovasos en lesiones ateroscleróticas de arterias coronarias humanas con diferentes grados de lesión en relación con la infiltración de células inflamatorias y moléculas del plasma. Pacientes y método. Se estudiaron 60 arterias coronarias procedentes de pacientes sometidos a operaciones de trasplante cardíaco. Por técnicas de histología convencional, inmunohistoquímica y análisis de imagen se analizaron y cuantificaron los marcadores celulares (célula muscular lisa, monocito/macrófago), la presencia de lípidos, de trombina/protrombina y los valores de expresión del factor de crecimiento endotelial de origen vascular (VEGF). Resultados. Se detectaron neovasos en lesiones avanzadas y se observó una correlación positiva con el grado de intrusión, la infiltración de monocitos/macrófagos y el contenido lipídico. Las células musculares lisas fueron las principales productoras de VEGF, tanto en la íntima como en la media de las lesiones avanzadas. En estas lesiones se observó colocalización de zonas con un alto contenido de trombina/protrombina, con células musculares en estado activado. Conclusiones. La presencia de neovasos en las lesiones de las arterias coronarias se correlaciona con el contenido de células inflamatorias, de material lipídico y de trombina/protrombina. La expresión de VEGF se asocia principalmente a las células musculares lisas, lo que indica un papel clave de estas células como moduladoras de las células endoteliales (AU)