Normal Human Coronary Arteries Research Articles

Abstract 14416: The Redox Effect of Stem-Cell Derived Extracellular Vesicles on Normal and Diabetic Human Coronary Artery Endothelial Cells

Introduction: Cardiovascular disease continues to be the leading cause of death globally, necessitating the study of novel therapeutic modalities. Following the lack of success with stem-cell based therapies, their extracellular vesicles (EVs) have emerged as a promising strategy. Hypothesis: We hypothesized that EVs will reduce oxidative stress in normal and diabetic human coronary artery endothelial cells (HCAEC). Methods: Human bone marrow-derived stem cells (HBMSC) were grown to passage 7 and subject to 24 hours of preconditioning by serum starvation media. EVs were then isolated via ultracentrifugation and submitted for LC-MS/MS for proteomics. Normal HCAEC and diabetic HCAEC (DM-HCAEC) were starved for 12 hours then treated with vehicle (1% DMSO in PBS) or EVs (1x108 particles/ml) for 6 hours. Following treatment, mitochondrial (mito) ROS using mitosox, total ROS using DCF-DA, and protein expression using Western blots were analyzed by 2-way ANOVA with Bonferroni multiple-comparisons correction. Results: A total of 473 EV proteins were identified, among which were antioxidants catalase, thioredoxin, peroxiredoxins, SOD1, and ferritin. EV-treatment reduced mito-ROS levels significantly in HCAEC, but not in DM-HCAEC. Basal (ambient) levels of total ROS in DM-HCAEC were significantly higher compared to HCAEC (p=0.0393), however, EV treatment reduced total ROS in both cell types significantly (p=0.002). Western blot analysis revealed that EV increased mitochondrial antioxidant MnSOD (SOD2) levels in HCAEC but not in DM-HCAEC. Catalase expression was unaffected by EV treatment in both cell types. Conclusions: EVs displayed differential effects on the mito-ROS, but reduced total ROS in both HCAEC and DM-HCAEC. EV-induced reduction in mito-ROS in non-diabetic HCAEC plausibly resulted from corresponding increase in MnSOD expression. Data from our ongoing studies will be presented at the meeting demonstrating whether the above changes in redox states by EVs correspond to differential proliferation, migration and angiogenesis in HCAEC vs. DM-HCAEC.

Lysophosphatidylcholine in phospholipase A2-modified LDL triggers secretion of angiopoietin 2

Background and aimsSecretory phospholipase A2 (PLA2) hydrolyzes LDL phospholipids generating modified LDL particles (PLA2-LDL) with increased atherogenic properties. Exocytosis of Weibel–Palade bodies (WPB) releases angiopoietin 2 (Ang2) and externalizes P-selectin, which both play important roles in vascular inflammation.Here, we investigated the effects of PLA2-LDL on exocytosis of WPBs. MethodsHuman coronary artery endothelial cells (HCAECs) were stimulated with PLA2- LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA2-LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice. ResultsExposure of HCAECs to PLA2-LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA2-LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA2-LDL also induced murine endothelial WPB exocytosis in blood vessels in and ex vivo, as evidenced by secretion of Ang2 in vivo, P-selectin translocation to plasma membrane in intact endothelial cells in thoracic artery and tracheal vessels, and reduced Ang2 staining in tracheal endothelial cells. Finally, in contrast to normal human coronary arteries, in which Ang2 was present only in the endothelial layer, at sites of advanced atherosclerotic lesions, Ang2 was detected also in the intima, media, and adventitia. ConclusionsOur studies reveal PLA2-LDL as a potent agonist of endothelial WPB exocytosis, resulting in increased secretion of Ang2 and translocation of P-selectin. The results provide mechanistic insight into PLA2-LDL-dependent promotion of vascular inflammation and atherosclerosis.

Label-free identification of human coronary atherosclerotic plaque based on a three-dimensional quantitative assessment of multiphoton microscopy images.

The rupture of coronary atherosclerotic plaque (CAP) and the resulting intracoronary thrombosis account for most acute coronary syndromes. Thus, the early identification and risk assessment of CAP is crucial for timely medical intervention. In this study, we propose a quantitative and label-free method for human CAP identification using multiphoton microscopy (MPM) and three-dimensional (3D) image analysis techniques. By detecting the intrinsic MPM signals, the microstructures of collagen and elastin fibers within normal and CAP-lesioned human coronary artery walls were imaged. Using a 3D gray level co-occurrence matrix method and 3D weighted vector summation algorithm, quantitative indicators that characterize the spatial texture and orientation features of the fibers were extracted. We demonstrate that these indicators show superior accuracy and repeatability over 2D texture features in CAP discrimination. Furthermore, by combining the 3D microstructural indicators, a support vector machine model that classifies CAP from the normal arterial wall with an accuracy of >97% was established. In conjunction with advances in multiphoton endoscopy, the proposed method shows great potential in providing a quantitative, label-free, and real-time tool for the early identification and risk assessment of CAP in the future.

Correlation between geometric parameters of the left coronary artery and hemodynamic descriptors of atherosclerosis: FSI and statistical study.

The hemodynamics conditioned by coronary geometry may play an important role in the creation of a pro-atherogenic environment in specific locations of the coronary tree. The aim of this study is to identify how several geometric parameters of the left coronary artery - cross-section areas, proximal left anterior descending artery length, angles between the branches and the septum, curvature and tortuosity - can be related with hemodynamic descriptors, using a computational fluid-structure interaction method. It is widely accepted that the hemodynamic indicators play an important role in identifying possible pro-atherogenic locations. A statistical study, using Pearson correlation coefficient and P value, was performed for a population study of 8 normal human left coronary arteries presenting right-dominant circulation. Within the study cases, arteries with high caliber (r = 0.88), high angles LMS-LAD (r = 0.49), LAD-LCx (r = 0.57) and LAD-Septum (r = 0.52), and high tortuosity LMS-LCx (r = 0.63) were correlated with a hemodynamic behavior propitious to plaque formation in the left anterior descending artery. In contrast, high proximal left anterior descending artery length (r = -0.41), high angle LMS-LCx (r = -0.59), high tortuosity LMS-LAD (r = -0.56) and LAD-LCx (r = -0.55) and high curvature of LMS (r = -0.60) and LCx (r = -0.56) can lead to non-favorable hemodynamic conditions for atheroma formation. Graphical abstract.

Imaging Cellular Structures of Atherosclerotic Coronary Arteries Using Circumferentially Scanning Micro-Optical Coherence Tomography Fiber Probe Ex Vivo

Development and progression of coronary atherosclerotic lesions is mediated by a number of cellular components that are not readily visualized using the current clinical investigation tools. Visualizing these cellular components in situ and in vivo may allow early detection of the vulnerable plaques, with implications for coronary artery disease therapy and for the prevention of acute myocardial infarction. In this paper, we have developed a fiber-optic micro-optical coherence tomography ( $\mu $ OCT) probe for intravascular use. We conducted ex vivo imaging experiments in normal swine aorta and human atherosclerotic coronary arteries and demonstrated that the fiber-probe-based $\mu $ OCT could delineate not only the layered structures of arterial wall but also the cellular-level anatomical structures of atherosclerotic plaques, including foam cells and smooth muscle cells. These results demonstrate the feasibility of intravascular $\mu $ OCT imaging.

Aortic calcified particles modulate valvular endothelial and interstitial cells.

Normal and calcified human valve cusps, coronary arteries, and aortae harbor spherical calcium phosphate microparticles of identical composition and crystallinity, and their role remains unknown. The objective was to examine the direct effects of isolated calcified particles on human valvular cells. Calcified particles were isolated from healthy and diseased aortae, characterized, quantitated, and applied to valvular endothelial cells (VECs) and interstitial cells (VICs). Cell differentiation, viability, and proliferation were analyzed. Particles were heterogeneous, differing in size and shape, and were crystallized as calcium phosphate. Diseased donors had significantly more calcified particles compared to healthy donors (P<.05), but there were no differences between the composition of the particles from healthy and diseased donors. VECs treated with calcified particles showed a significant decrease in CD31 and VE-cadherin and an increase in von Willebrand factor expression, P<.05. There were significantly increased α-SMA and osteopontin in treated VICs (P<.05), significantly decreased VEC and VIC viability (P<.05), and significantly increased number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive VECs (P<.05) indicating apoptosis when treated with the calcified particles. Isolated calcified particles from human aortae are not innocent bystanders but induce a phenotypical and pathological change of VECs and VICs characteristic of activated and pathological cells. Therapy tailored to reduce these calcified particles should be investigated.

Tumor-targeted and pH-controlled delivery of doxorubicin using gold nanorods for lung cancer therapy.

BackgroundIn lung cancer, the efficacy of conventional chemotherapy is limited due to poor drug accumulation in tumors and nonspecific cytotoxicity. Resolving these issues will increase therapeutic efficacy.MethodsGNR-Dox-Tf-NPs (gold nanorod-doxorubicin-transferrin-nanoparticles) were prepared by different chemical approaches. The efficacy of these nanoparticles was carried out by cell viability in lung cancer and primary coronary artery smooth muscle cells. The receptor-mediated endocytosis studies were done with human transferrin and desferrioxamine preincubation. The GNR-Dox-Tf nanoparticles induced apoptosis, and DNA damage studies were done by Western blot, H2AX foci, and comet assay.ResultsWe developed and tested a gold nanorod-based multifunctional nanoparticle system (GNR-Dox-Tf-NP) that carries Dox conjugated to a pH-sensitive linker and is targeted to the transferrin receptor overexpressed in human lung cancer (A549, HCC827) cells. GNR-Dox-Tf-NP underwent physicochemical characterization, specificity assays, tumor uptake studies, and hyperspectral imaging. Biological studies demonstrated that transferrin receptor-mediated uptake of the GNR-Dox-Tf-NP by A549 and HCC827 cells produced increased DNA damage, apoptosis, and cell killing compared with nontargeted GNR-Dox-NP. GNR-Dox-Tf-NP-mediated cytotoxicity was greater (48% A549, 46% HCC827) than GNR-Dox-NP-mediated cytotoxicity (36% A549, 39% HCC827). Further, GNR-Dox-Tf-NP markedly reduced cytotoxicity in normal human coronary artery smooth muscle cells compared with free Dox.ConclusionThus, GNR-Dox-Tf nanoparticles can selectively target and deliver Dox to lung tumor cells and alleviate free Dox-mediated toxicity to normal cells.

Endosomes: guardians against [Ru(Phen)3]2+ photo-action in endothelial cells during in vivo pO2 detection?

Phototoxicity is a side-effect of in vitro and in vivo oxygen partial pressure (pO2) detection by luminescence lifetime measurement methods. Dichlorotris(1,10-phenanthroline)-ruthenium(ii) hydrate ([Ru(Phen)3]2+) is a water soluble pO2 probe associated with low phototoxicity, which we investigated in vivo in the chick's chorioallantoic membrane (CAM) after intravenous or topical administration and in vitro in normal human coronary artery endothelial cells (HCAEC). In vivo, the level of intravenously injected [Ru(Phen)3]2+ decreases within several minutes, whereas the maximum of its biodistribution is observed during the first 2 h after topical application. Both routes are followed by convergence to almost identical "intra/extra-vascular" levels of [Ru(Phen)3]2+. In vitro, we observed that [Ru(Phen)3]2+ enters cells via endocytosis and is then redistributed. None of the studied conditions induced modification of lysosomal or mitochondrial membranes without illumination. No nuclear accumulation was observed. Without illumination [Ru(Phen)3]2+ induces changes in endoplasmic reticulum (ER)-to-Golgi transport. The phototoxic effect of [Ru(Phen)3]2+ leads to more marked ultrastructural changes than administration of [Ru(Phen)3]2+ only (in the dark). These could lead to disruption of Ca2+ homeostasis accompanied by mitochondrial changes or to changes in secretory pathways. In conclusion, we have demonstrated that the intravenous injection of [Ru(Phen)3]2+ into the CAM model mostly leads to extracellular localization of [Ru(Phen)3]2+, while its topical application induces intracellular localization. We have shown in vivo that [Ru(Phen)3]2+ induces minimal photo-damage after illumination with light doses larger by two orders of magnitude than those used for pO2 measurements. This low phototoxicity is due to the fact that [Ru(Phen)3]2+ enters endothelial cells via endocytosis and is then redistributed towards peroxisomes and other endosomal and secretory vesicles before it is eliminated via exocytosis. Cellular response to [Ru(Phen)3]2+, survival or death, depends on its intracellular concentration and oxidation-reduction properties.

Synthetic prostacyclin agonist, ONO1301, enhances endogenous myocardial repair in a hamster model of dilated cardiomyopathy: A promising regenerative therapy for the failing heart

Remodeling of the left ventricle (LV) in idiopathic dilated cardiomyopathy (IDCM) is known to be associated with multiple pathologic changes that endogenous factors, such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), protect against. Although a clinically relevant delivery method of these factors has not been established, ONO1301, a synthetic prostacyclin agonist, has been shown to upregulate multiple cardioprotective factors, including HGF and VEGF, invivo. We thus hypothesized that ONO1301 may reverse LV remodeling in the DCM heart. ONO1301 dose-dependently added to the normal human dermal fibroblasts and human coronary artery smooth muscle cells invitro, to measure the expression of HGF, VEGF, stromal cell-derived factor (SDF)-1, and granulocyte-colony stimulating factor (G-CSF), assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. δ-Sarcoglycan-deficient J2N-k hamsters, which is an established DCM model, were treated by epicardial implantation of an atelocollagen sheet with or without ONO1301 immersion or sham operation. ONO1301 dose-dependently upregulated expression of these 4 factors invitro. ONO1301 treatment, which induced dominant elevation of ONO1301 levels for 2 weeks, significantly preserved cardiac performance and prolonged survival compared with the other groups. This treatment significantly upregulated expressions of cardioprotective factors and was associated with increased capillaries, attenuated fibrosis, and upregulation of α-sarcoglycan in the DCM heart. ONO1301 atelocollagen-sheet implantation reorganized cytoskeletal proteins, such as α-sarcoglycan, increased capillaries, reduced fibrosis, and was associated with upregulated expression of multiple cardioprotective factors, leading to preservation of cardiac performance and prolongation of survival in the δ-sarcoglycan-deficient DCM hamster.

Cataloguing the geometry of the human coronary arteries: A potential tool for predicting risk of coronary artery disease

Background The non-uniform distribution of atherosclerosis in the human vasculature suggests that local fluid dynamics or wall mechanics may be involved in atherogenesis. Thus certain aspects of vascular geometry, which mediates both fluid dynamics and wall mechanics, might be risk factors for coronary atherosclerosis. Cataloguing the geometry of normal human coronary arteries and its variability is a first step toward identifying specific geometric features that increase vascular susceptibility to the disease. Methods Images of angiographically normal coronary arteries, including 32 left anterior descending (LAD) and 35 right coronary arteries (RCA), were acquired by clinical biplane cineangiography from 52 patients. The vessel axes in end diastole were reconstructed and geometric parameters that included measures of curvature, torsion and tortuosity were quantified for the proximal, middle and distal segments of the arteries. Results Statistical analysis shows that (1) in the LAD, curvature, torsion and tortuosity are generally highest in the distal portion, (2) in the RCA, these parameters are smallest in the middle segment, (3) the LAD exhibits significant higher torsion than the RCA ( P < 0.005), and (4) > 80% of the variability of coronary arterial geometry can be expressed in terms of two factors, one dominated by the curvature measures and tortuosity, and the other emphasizing the torsion parameters. Conclusions This study has comprehensively documented the normal arterial geometry of the LAD and RCA in end diastole. This information may be used to guide the identification of geometric features that might be atherogenic risk factors.

Receptors for the anaphylatoxins C3a and C5a are expressed in human atherosclerotic coronary plaques

The anaphylatoxins C3a and C5a are potent chemotactic and pro-inflammatory peptides that are released during complement activation, and recent clinical work have suggested them a role in acute coronary events. Here we studied whether human coronary plaques express anaphylatoxin receptors C3aR and C5aR, i.e. whether they have the potential to respond to anaphylatoxins. For this purpose, both normal ( n = 14) and atherosclerotic ( n = 20) human coronary artery samples were collected for histological and PCR analyses. Immunohistochemistry demonstrated that in atherosclerotic, but not in normal intimas, C3aR and C5aR were present. Consistently, PCR analysis showed that the expression of both receptors was >5-fold increased in the atherosclerotic plaques ( p < 0.01). Double immunofluorescence stainings revealed that in the plaques the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR and a small fraction of them also expressed C3aR, the mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. In conclusion, the presence of receptors for anaphylatoxins in human coronary plaques suggests that anaphylatoxins activate coronary plaques, and points the complement system as a potential therapeutic target in attempts to stabilize them.

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1β, tumor necrosis factor-α, interferon-β, interferon-γ, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.

Flow parameters in normal left coronary artery tree. Implication to atherogenesis

The dominant haemodynamic flow parameters of wall pressure (WP), wall shear stress (WSS), molecular viscosity and the spatial gradients: wall pressure gradient (WPG) and wall shear stress gradient (WSSG) along the normal human left coronary artery (LCA) tree are numerically analyzed in relation to atheronegenesis. The LCA tree includes the left main coronary artery, the left anterior descending branch, the left circumflex branch and their major branches. Spatial differentiation indicates that low values of WP (locally), WPG, WSS, WSSG and high molecular viscosity appear opposite flow dividers and this probably correlates to atherosclerosis localization.

Spatial and phasic oscillation of non-Newtonian wall shear stress in human left coronary artery bifurcation: an insight to atherogenesis

To investigate the wall shear stress oscillation in a normal human left coronary artery bifurcation computational model by applying non-Newtonian blood properties and phasic flow. The three-dimensional geometry of the investigated model included the left main coronary artery along with its two main branches, namely the left anterior descending and the left circumflex artery. For the computational analyses a pulsatile non-Newtonian flow was applied. To evaluate the cyclic variations in wall shear stress, six characteristic time-points of the cardiac cycle were selected. The non-Newtonian wall shear stress variation was compared with the Newtonian one. The wall shear stress varied remarkably in time and space. The flow divider region encountered higher wall shear stress values than the lateral walls throughout the entire cardiac cycle. The wall shear stress exhibited remarkably lower and oscillatory values in systole as compared with that in diastole in the entire bifurcation region, especially in the lateral walls. Although the Newtonian wall shear stress experienced consistently lower values throughout the entire cardiac cycle than the non-Newtonian wall shear stress, the general pattern of lower wall shear stress values at the lateral walls, particularly during systole, was evident regardless of the blood properties. The lateral walls of the bifurcation, where low and oscillating wall shear stress is observed, are more susceptible to atherosclerosis. The systolic period, rather than the diastolic one, favors the development and progression of atherosclerosis. The blood viscosity properties do not seem to qualitatively affect the spatial and temporal distribution of the wall shear stress.

Frontiers in Cardiovascular Magnetic Resonance

Cardiovascular magnetic resonance (MR) is emerging as a multipurpose imaging modality for the assessment of cardiovascular disease in general and ischemic heart disease in particular. Currently, the pace of innovation is rapid, and the modality is changing from one that is used primarily as a research tool to one that is increasingly used in routine clinical practice. The process of innovation includes not only improvements in scanner hardware, such as coil and gradient technology, and the development of new contrast agents but also the development of novel pulse sequences. The concept of the pulse sequence, in which programming changes at the scanner can lead to fundamental changes in activating tissue, is unique to MR and gives this modality the potential to assess a vast number of biological parameters. Cardiovascular MR promises to play an important clinical and investigational role in both vascular and cardiac systems. Current and potential future applications of cardiovascular MR will be discussed with a particular focus on ischemic heart disease. Multidetector-row computed tomography, another promising and complementary noninvasive imaging technology, will be discussed briefly in relation to cardiovascular MR for the assessment of atherothrombotic disease. ### Nomenclature and Evolving Imaging Assessment Atherothrombosis is a systemic or multiterritory arterial disease that primarily affects the large- and medium systemic arteries, including the aorta and the carotid, coronary, and peripheral arteries. Although the epicardial coronary arteries appear to be the most susceptible to atherothrombosis,1,2 intramyocardial arteries are relatively resistant. The concept of multiterritory atherothrombosis has been addressed in 2 large studies of symptomatic patients that showed that at entry into the studies, 3% to 8% had symptomatic atherothrombotic disease in all 3 main arterial districts and 23% to 32% had disease in 2 districts.3,4 From a structural point of view, the 4 main components of the atherothrombotic plaques are as follows: (1) fibrocellular, …

Wall pressure gradient in normal left coronary artery tree

The three-dimensional wall pressure gradient (WPG) of the normal human left coronary artery (LCA) tree is quantitatively analysed. A model LCA tree, based on averaged human data set extracted from angiographies was adopted for finite-element analysis. The LCA tree includes the left main coronary artery (LMCA), the left anterior descending (LAD), the left circumflex artery (LCxA) and their major branches. The WPG is calculated using 44,452 nodes throughout the tree extension. The governing flow equations were solved using a validated numerical code. WPG as well as wall shear stress gradient (WSSG) were calculated at all available bifurcation regions. In proximal LCA tree regions where atherosclerosis frequently occurs, low WPG appears. At distal segments, WPG increases substantially due to increased velocity resulted from increased vessel tapering. Low WPG occurs at bifurcations in regions opposite the apexes, which are anatomic sites predisposed for atherosclerotic development. Endothelial cells respond to the combined effects of locally low WPG and low WSSG and provide a mechanism promoting atherosclerosis. This computational work determines probably for the first time the topography of the WPG in the normal human LCA tree. Spatial WPG differentiation indicates that locally low values of this physical parameter probably correlate to atherosclerosis localization.

Wall shear stress in normal left coronary artery tree

Despite the fact that the role of wall shear stress (WSS) as a local mechanical factor in atherogenesis is well established, its distribution over the entire normal human left coronary artery (LCA) tree has not yet been studied. A three-dimensional computer generated model of the epicardial LCA tree, based on averaged human data set extracted from angiographies, was adopted for finite-element analysis of the Navier–Stokes flow equations treating blood as non-Newtonian fluid. The LCA tree includes the left main coronary artery (LMCA), the left anterior descending (LAD), the left circumflex artery (LCxA) and their major branches. In proximal LCA tree regions where atherosclerosis frequently occurs, low WSS appears. Low WSS regions occur at bifurcations in regions opposite the flow dividers, which are anatomic sites predisposed for atherosclerotic development. On the LMCA bifurcation, at regions opposite to the flow divider, dominant low WSS values occur ranging from 0.75 to 2.25 N/m 2. High WSS values are encountered at all flow dividers. This work determines, probably for the first time, the topography of the WSS in the entire normal human LCA epicardial tree. It is also the first work determining the spatial WSS differentiation between proximal and distal normal human LCA parts. The haemodynamic analysis of the entire epicardial LCA tree further verifies the implications of the WSS in atherosclerosis mechanisms.

Expression, localisation and function of ACE and chymase in normal and atherosclerotic human coronary arteries

The expression, localisation and function of enzymes responsible for the local formation of angiotensin II in atherosclerotic and non-atherosclerotic human coronary arteries were studied. Human epicardial coronary arteries expressed mRNA for both ACE and chymase. Immunohistochemical studies revealed that ACE was localised to the vascular endothelium, and to a lesser extent the medial smooth muscle cells, in both large and small arteries. Chymase was detected in both types of vessel but was shown to be associated with mast cells. The contractions to angiotensin I in large arteries were inhibited only by a combination of quinaprilat and soyabean trypsin inhibitor. In the intramyocardial arteries the response to angiotensin I was markedly inhibited in the presence of chymostatin. These findings demonstrate that a dual pathway for the synthesis of angiotensin II is active in diseased and non-diseased coronary arteries.

Role of β 2 Adrenergic Receptors in Human Atherosclerotic Coronary Arteries

Adrenergic regulation of coronary vasomotion is balanced between alpha1-adrenergic-mediated (alpha1-AR) constriction and beta2-adrenergic-mediated (beta2-AR) relaxation. This study aimed at assessing the role of beta2-ARs in normal, mildly atherosclerotic, and stenotic human coronary arteries. During intracoronary (IC) infusion of increasing doses of the beta2-AR agonist salbutamol (0.15, 0.3, and 0.6 mug/min) and the endothelial vasodilator acetylcholine (1, 3, and 10 microg/min), we measured (1) changes in lumen diameter (LD) by quantitative coronary angiography in 34 normal, 55 mildly atherosclerotic, and 42 stenotic coronary artery segments and (2) changes in average peak velocity (APV) and coronary blood flow (CBF) with the use of Doppler flow wire in 11 normal, 10 mildly atherosclerotic, and 11 stenotic coronary arteries. In 6 of 11 stenotic coronary arteries, the protocol was repeated after an IC bolus (12 microg/kg) of the alpha-adrenergic blocker phentolamine. In 6 of 11 normal coronary arteries, the protocol was repeated after an IC infusion (60 micromol/min) of N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide inhibitor. Neither salbutamol IC infusion nor acetylcholine significantly changed heart rate or blood pressure, whereas L-NMMA slightly increased blood pressure. In normal coronary arteries, salbutamol increased LD (LD max %: 11+/-2, P<0.05), APV (APV max %: 53+/-17, P<0.05), and CBF (CBF max %: 57+/-17, P<0.05), whereas L-NMMA caused a blunted APV (APV max %: 27+/-6, P<0.05) and CBF (CBF max %: 29+/-6, P<0.05) response to salbutamol. In mildly atherosclerotic coronary arteries, the salbutamol increase in LD (LD max %: 10+/-2, P<0.05), APV (APV max %: 33+/-12, P<0.05), and CBF (CBF max %: 37+/-12, P<0.05) was preserved. In stenotic coronary arteries, salbutamol induced a paradoxical reduction in LD (LD max %: -6+/-2, P<0.05), APV (APV max %: -15+/-9, P<0.05), and CBF (CBF max %: -15+/-6, P<0.05), which was no longer observed after phentolamine. Acetylcholine increased LD (LD max %: 14+/-3, P<0.05), APV (APV max %: 61+/-20, P<0.05), and CBF (CBF max %: 67+/-19, P<0.05) in normal coronary arteries. In mildly atherosclerotic coronary arteries, acetylcholine induced a significant reduction in LD (LD max %: -15+/-2, P<0.05) and no changes in APV (APV max %: -6+/-13, P=NS) and CBF (CBF max %: -10+/-13, P=NS). In stenotic coronary arteries, acetylcholine significantly reduced LD (LD max %: -15+/-3, P<0.05), APV (APV max %: -15+/-9, P<0.05), and CBF (CBF max %: -15+/-6, P<0.05). In severely atherosclerotic coronary arteries, beta2-adrenergic vasodilatation is impaired, and this might contribute to alter the vasomotor balance, further precipitating myocardial ischemia during sympathetic activation.

AIF-1 expression modulates proliferation of human vascular smooth muscle cells by autocrine expression of G-CSF.

Allograft inflammatory factor-1 (AIF-1) is associated with vascular smooth muscle cell (VSMC) activation and vascular injury. The purpose of this study was to characterize the molecular mechanism of AIF-1 growth-enhancing effects in human VSMC. Primary human VSMCs were stably transduced with AIF-1 retrovirus (RV). Impact on cell growth was evaluated by the increase in cell number, and the effects on gene expression were determined by cDNA microarray analysis. AIF-RV overexpressing cells grew significantly more rapidly than empty-RV control cells in growth medium and serum-reduced medium (P<0.01 and 0.02, respectively). cDNA microarray analysis and Western blotting on serum-starved AIF-1-transduced VSMCs identified increased mRNA expression of several cell cycle proteins and, surprisingly, the cytokine G-CSF. Addition of G-CSF caused a 75% increase in proliferation of VSMCs in the absence of serum growth factors. The proliferative effects of AIF-1 were abrogated by neutralizing antibodies to G-CSF (P<0.05), and AIF-1-transduced VSMCs are chemotactic for human monocytes. Increased expression of G-CSF and colocalization with AIF-1 positive cells were seen in diseased, not normal human coronary arteries. This study indicates that AIF-1 enhances VSMC growth by autocrine production of G-CSF, and AIF-1 expression may influence VSMC-inflammatory cell communication.