Human Coronary Artery Segments Research Articles

Pharmacology of erenumab in human isolated coronary and meningeal arteries: Additional effect of gepants on top of a maximum effect of erenumab.

Multiple drugs targeting the calcitonin gene-related peptide (CGRP) receptor have been developed for migraine treatment. Here, the effect of the monoclonal antibody erenumab on CGRP-induced vasorelaxation was investigated in human isolated blood vessels, as well as the effect of combining erenumab with the small molecule drugs, namely rimegepant, olcegepant, or sumatriptan. Concentration-response curves to CGRP, adrenomedullin or pramlintide were constructed in human coronary artery (HCA) and human middle meningeal artery (HMMA) segments, incubated with or without erenumab and/or olcegepant. pA2 or pKb values were calculated to determine the potency of erenumab in both tissues. To study whether acutely acting antimigraine drugs exerted additional CGRP-blocking effects on top of erenumab, HCA segments were incubated with a maximally effective concentration of erenumab (3μM), precontracted with KCl and exposed to CGRP, followed by rimegepant, olcegepant, or sumatriptan in increasing concentrations. Erenumab shifted the concentration-response curve to CGRP in both vascular tissues. However, in HCA, the Schild plot slope was significantly smaller than unity, whereas this was not the case in HMMA, indicating different CGRP receptor mechanisms in these tissues. In HCA, rimegepant, olcegepant and sumatriptan exerted additional effects on CGRP on top of a maximal effect of erenumab. Gepants have additional effects on top of erenumab for CGRP-induced relaxation and could be effective in treating migraine attacks in patients already using erenumab as prophylaxis.

Are ovine and porcine carotid arteries equivalent animal models for experimental cardiac surgery: A quantitative histological comparison

BackgroundCoronary artery bypass grafting (CABG) is a common cardiac surgery. Manufacturing small-diameter (2–5 mm) vascular grafts for CABG is important for patients who lack first-choice autologous arterial, or venous conduits. Ovine and porcine common carotid arteries (CCAs) are used as large animal models for in vivo testing of newly developed tissue-engineered arterial grafts. It is unknown to what extent these models are interchangeable and whether the left and right arteries of the same subjects can be used as experimental controls. Therefore, we compared the microscopic structure of paired left and right ovine and porcine CCAs in the proximodistal direction and compared these animal model samples to samples of human coronary arteries (CAs) and human internal thoracic arteries (ITAs). MethodsWe compared the histological composition of whole CCAs of sheep (n = 22 animals) with whole porcine CCAs (n = 21), segments of human CAs (n = 21), and human ITAs (n = 21). Using unbiased sampling and stereological methods, we quantified the fractions of elastin, total collagen, type I collagen, type III collagen, smooth muscle actin (SMA) and chondroitin sulfate (CS) A, B, and C. We also quantified the densities and distributions of nuclear profiles, nervi vasorum and vasa vasorum as well as the thickness of the intima-media and total wall thickness. ResultsThe differences between the paired samples of left and right CCAs in sheep were substantially greater than the differences in laterality in porcine CCAs. The right ovine CCAs had a smaller fraction of elastin (p < 0.001), greater fraction of SMA (p < 0.01), and greater intima-media thickness (p < 0.001) than the paired left side CCAs. In pigs, the right CCAs had a greater fraction of elastin (p < 0.05) and a greater density of vasa vasorum in the media (p < 0.001) than the left-side CCAs. The fractions of elastin and CS decreased and the fraction of SMA increased in the proximodistal direction in both the ovine (p < 0.001) and porcine (p < 0.001) CCAs. Ovine CCAs had a muscular phenotype along their entire length, but porcine CCAs were elastic-type arteries in the proximal segments but muscular type arteries in middle and distal segments. The CCAs of both animals differed from the human CAs and ITAs in most parameters, but the ovine CCAs had a comparable fraction of elastin and CS to human ITAs. ConclusionsFrom a histological point of view, ovine and porcine CCAs were not equivalent in most quantitative parameters to human CAs and ITAs. Left and right ovine CCAs did not have the same histological composition, which is limiting for their mutual equivalence as sham-operated controls in experiments. These differences should be taken into account when designing and interpreting experiments using these models in cardiac surgery. The complete morphometric data obtained by quantitative evaluation of arterial segments were provided to facilitate the power analysis necessary for justification of the minimum number of samples when planning further experiments. The middle or distal segments of ovine and porcine CCAs remain the most realistic and the best characterized large animal models for testing artificial arterial CABG conduits.

Promyelocytic leukemia protein promotes the phenotypic switch of smooth muscle cells in atherosclerotic plaques of human coronary arteries.

Promyelocytic leukemia protein (PML) is a constitutive component of PML nuclear bodies (PML-NBs), which function as stress-regulated SUMOylation factories. Since PML can also act as a regulator of the inflammatory and fibroproliferative responses characteristic of atherosclerosis, we investigated whether PML is implicated in this disease. Immunoblotting, ELISA and immunohistochemistry showed a stronger expression of PML in segments of human atherosclerotic coronary arteries and sections compared with non-atherosclerotic ones. In particular, PML was concentrated in PML-NBs from α-smooth muscle actin (α-SMA)-immunoreactive cells in plaque areas. To identify possible functional consequences of PML-accumulation in this cell type, differentiated human coronary artery smooth muscle cells (dHCASMCs) were transfected with a vector containing the intact PML-gene. These PML-transfected dHCASMCs showed higher levels of small ubiquitin-like modifier (SUMO)-1-dependent SUMOylated proteins, but lower levels of markers for smooth muscle cell (SMC) differentiation and revealed more proliferation and migration activities than dHCASMCs transfected with the vector lacking a specific gene insert or with the vector containing a mutated PML-gene coding for a PML-form without SUMOylation activity. When dHCASMCs were incubated with different cytokines, higher PML-levels were observed only after interferon γ (IFN-γ) stimulation, while the expression of differentiation markers was lower. However, these phenotypic changes were not observed in dHCASMCs treated with small interfering RNA (siRNA) suppressing PML-expression prior to IFN-γ stimulation. Taken together, our results imply that PML is a previously unknown functional factor in the molecular cascades associated with the pathogenesis of atherosclerosis and is positioned in vascular SMCs (VSMCs) between upstream IFN-γ activation and downstream SUMOylation.

Quantitative proteomics reveals the regulatory networks of circular RNA BTBD7_hsa_circ_0000563 in human coronary artery.

BackgroundBTBD7_hsa_circ_0000563, which is located on chromosome 14, contains conserved binding sites with miR‐155/130a and RNA‐binding proteins according to bioinformatic prediction. We investigated the association of BTBD7_hsa_circ_0000563 expression in coronary artery segments with atherosclerotic stenosis and identified the proteome‐wide BTBD7_hsa_circ_0000563‐regulated proteins in human coronary artery.MethodsThe atherosclerotic grade and extent in coronary artery segments were determined by hematoxylin and eosin staining. BTBD7_hsa_circ_0000563 expression in eight coronary artery segments from one patient was quantified by RT‐qPCR assay. A proteomic approach was adopted to reveal significant differences in protein expression between among four groups differing in their BTBD7_hsa_circ_0000563 expression levels.ResultsThe RT‐qPCR assay revealed that coronary artery segments with severe atherosclerotic stenosis had significantly low BTBD7_hsa_circ_0000563 levels. The proteomic analysis identified 49 differentially expressed proteins among the segment groups with different BTBD7_hsa_circ_0000563 expression levels, of which 10 were downregulated and 39 were upregulated with increases in the BTBD7_hsa_circ_0000563 level. The 10 downregulated proteins were P61626 (LYSC_HUMAN), P02760 (AMBP_HUMAN), Q02985 (FHR3_HUMAN), P01701 (LV151_HUMAN), P06312(KV401_HUMAN), P01624 (KV315_HUMAN), P13671 (CO6_HUMAN), P01700(LV147_HUMAN), Q9Y287(ITM2B_HUMAN), and A0A075B6I0 (LV861_HUMAN). The top 10 upregulated proteins were Q92552 (RT27_HUMAN), Q9UJY1(HSPB8_HUMAN), Q9Y235(ABEC2_HUMAN), P19022 (CADH2_HUMAN), O43837(IDH3B_HUMAN), Q9H479(FN3K_HUMAN), Q9UM22(EPDR1_HUMAN), P48681(NEST_HUMAN), Q9NRP0(OSTC_HUMAN), and Q15628(TRADD_HUMAN).ConclusionBTBD7_hsa_circ_0000563 is involved in the atherosclerotic changes in human coronary artery segments. Verification, mechanistic, and function studies are needed to confirm whether patients with coronary artery disease would benefit from such personalized medicine in the future.

Histological mapping of porcine carotid arteries — An animal model for the assessment of artificial conduits suitable for coronary bypass grafting in humans

BackgroundUsing animal models in experimental medicine requires mapping of their anatomical variability. Porcine common carotid arteries (CCA) are often preferred for the preclinical testing of vascular grafts due to their anatomical and physiological similarity to human small-diameter arteries. Comparing the microscopic structure of animal model organs to their human counterparts reveals the benefits and limitations of translational medicine. MethodsUsing quantitative histology and stereology, we performed an extensive mapping of the regional proximodistal differences in the fractions of elastin, collagen, and smooth muscle actin as well as the intima-media and wall thicknesses among 404 segments (every 1 cm) of porcine CCAs collected from male and female pigs (n = 21). We also compared the microscopic structure of porcine CCAs with segments of human coronary arteries and one of the preferred arterial conduits used for the coronary artery bypass grafting (CABG), namely, the internal thoracic artery (ITA) (n = 21 human cadavers). ResultsThe results showed that the histological structure of left and right porcine CCA can be considered equivalent, provided that gross anatomical variations of the regular branching patterns are excluded. The proximal elastic carotid (51.2% elastin, 4.2% collagen, and 37.2% actin) transitioned to more muscular middle segments (23.5% elastin, 4.9% collagen, 54.3% actin) at the range of 2–3 centimeters and then to even more muscular distal segments (17.2% elastin, 4.9% collagen, 64.0% actin). The resulting morphometric data set shows the biological variability of the artery and is made available for biomechanical modeling and for performing a power analysis and calculating the minimum number of samples per group when planning further experiments with this widely used large animal model. ConclusionsComparison of porcine carotids with human coronary arteries and ITA revealed the benefits and the limitations of using porcine CCAs as a valid model for testing bioengineered small-diameter CABG vascular conduits. Morphometry of human coronary arteries and ITA provided more realistic data for tailoring multilayered artificial vascular prostheses and the ranges of values within which the conduits should be tested in the future. Despite their limitations, porcine CCAs remain a widely used and well-characterized large animal model that is available for a variety of experiments in vascular surgery.

ZFYVE21 is a Novel Complement-Induced Rab5 Effector Protein Mediating Cardiac Allograft Vasculopathy

Purpose Complement promotes vascular inflammation in transplant organ rejection, a process leading to cardiac allograft vasculopathy. In this study, we identify a new Rab5 effector protein, ZFYVE21, that mediates this process. We elucidate a ZFYVE21-SMURF2-pAkt signaling axis which activates non-canonical NF-kB, and we define the clinical relevance of this sequence. Methods We developed FACS-assisted protocols enabling proteomic analysis of MAC+Rab5+ endosomes and semi-quantitative analyses of the phosphoinositide content of these structures. A humanized mouse model incorporating human coronary artery segments and human lymphoid cells was employed to define a role for ZFYVE21-associated signaling in cardiac allograft vasculopathy. Software-assisted analyses of human transplant biopsies analyzing ZFYVE21 colocalization was performed using a computer algorithm we helped to developed in-house. Results In response to deposition of membrane attack complexes (MAC) on endothelial cells (EC), ZFYVE21 is post-translationally stabilized via recruitment to MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of the phosphoinositide content of MAC+Rab5+ endosomes with miltefosine reduces ZFYVE21 induction, EC activation, and MAC-induced allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC- but not ligand-induced non-canonical NF-κB and is a biomarker for complement-mediated endothelial signaling in transplant biopsies with antibody-mediated rejection. Conclusion Our data identifies ZFYVE21 as a novel Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway as a drug target and biomarker for allograft vasculopathy.

Development of a 3 French Dual-Frequency Intravascular Ultrasound Catheter

Coronary plaque morphology, including plaque size and fibrous cap thickness, is thought to contribute to the risk of plaque rupture and future cardiac events. Dual-frequency intravascular ultrasound has been proposed as a possible technique to visualize both large-scale features and superficial detail of coronary plaque; however, it has not been found to be feasible within the constraints of a clinically functional intravascular ultrasound catheter. In this study, we describe the design and fabrication of a dual-frequency catheter using a bidirectional transducer stack with center frequencies of approximately 30 and 80 MHz. We describe how the high-frequency transducer achieves significantly improved axial and lateral resolution (16 and 120 µm, respectively, vs. 50 and 220 µm) at the expense of penetration depth. Finally, imaging of ex vivo human coronary artery segments reveals that the catheter can provide complementary images of the deeper arterial wall and superficial plaque features.

P1777Differences in lectin-like oxidized low density lipoprotein receptor-1 expession in various segments of human coronary arteries

P1777Differences in lectin-like oxidized low density lipoprotein receptor-1 expession in various segments of human coronary arteries

Ultrafast optical-ultrasonic system and miniaturized catheter for imaging and characterizing atherosclerotic plaques in vivo

Atherosclerotic coronary artery disease (CAD) is the number one cause of death worldwide. The majority of CAD-induced deaths are due to the rupture of vulnerable plaques. Accurate assessment of plaques is crucial to optimize treatment and prevent death in patients with CAD. Current diagnostic techniques are often limited by either spatial resolution or penetration depth. Several studies have proved that the combined use of optical and ultrasonic imaging techniques increase diagnostic accuracy of vulnerable plaques. Here, we introduce an ultrafast optical-ultrasonic dual-modality imaging system and flexible miniaturized catheter, which enables the translation of this technology into clinical practice. This system can perform simultaneous optical coherence tomography (OCT)-intravascular ultrasound (IVUS) imaging at 72 frames per second safely in vivo, i.e., visualizing a 72 mm-long artery in 4 seconds. Results obtained in atherosclerotic rabbits in vivo and human coronary artery segments show that this ultrafast technique can rapidly provide volumetric mapping of plaques and clearly identify vulnerable plaques. By providing ultrafast imaging of arteries with high resolution and deep penetration depth simultaneously, this hybrid IVUS-OCT technology opens new and safe opportunities to evaluate in real-time the risk posed by plaques, detect vulnerable plaques, and optimize treatment decisions.

Abstract 417: Endosome-Based Activation of Non-Canonical NF-kB Signaling by Complement Membrane Attack Complexes

Using sera from transplant patients with high-titer panel reactive antibody (PRA), we previously described a model for studying the effects of alloantibody binding and complement activation on human endothelial cells (EC). With this model we found that complement membrane attack complexes (MAC) induced rapid post-translational stabilization of NF-kB-inducing kinase (NIK), the activator of non-canonical NF-kB signaling, which was required for inflammatory changes in cultured human EC and for vasculopathic changes in human coronary artery segments implanted into immunodeficient mouse hosts. To elucidate the mechanism of NIK stabilization, we performed a genome-wide siRNA screen and discovered a novel, TRAF3-independent signaling mechanism in which MAC induces NIK stabilization on endosomes. MAC is internalized by EC in a clathrin-, AP2-, and dynamin-dependent mechanism into Rab5+ endosomes. Activated Akt is recruited in a Rab5-dependent manner to the same endosomes which then bind and stabilize NIK and subsequently recruit and activate IKK-alpha, the catalytic activator of p52/RelB complexes, the non-canonical form of NF-kB. Pharmacological inhibition of MAC endocytosis blocked this pathway both in cultured EC and in human coronary artery xenografts. These data reveal a novel endosome-based signaling cascade for activating non-canonical NF-kB and identify new targets for inhibiting complement-mediated tissue injury such as chronic antibody-mediated rejection and cardiac allograft vasculopathy.

Abstract 416: IDL-Apolipoprotein E Isoform Navigates Human Coronary Artery Tone

Background: Apolipoprotein E4/E4 is the most significant risk factor known to cause Alzheimer dementia [1], but also crucial in atherogenesis as pathological correlative of coronary heart disease. The present investigation elucidates the role of IDLapoE isoforms in flow-dependent vascular reactivity. Methods: Isometric tension, membrane potential and cyclic nucleotide concentrations were measured in segments of human coronary arteries from heart transplantation. Results: The results of the mechanical recording are summarized in the Table. Upon a basal flow rate of 3 mL/min, the initial tension showed a graded course in normal preparations: cooled IDL (10 mg/dL) enhanced mechanical force (+0.8%), IDLapoE4/E4 (+9.5%) and IDLapoE0/E0 (from an apoE ‘knockout man’) (+19.1%) as compared to the controls in Krebs solution. Flow-dependent relaxation was impaired by 20% in apoE4/E4 solution, whereas apoE0/E0 caused a flow-dependent contraction by 85%. Endothelium-denuded segments had basically a higher tone on grounds of an impaired NO release. Membrane potentials reflected tension: hyperpolarization of the VSMCs preceded relaxation, depolarization preceded contraction. For the first time in literature, we report on a chemomechanical coupling curve. Conclusion: IDLapoE4/E4 impaired flow-dependent vasodilatation and was only exceeded in its deleterious effect by vasoconstrictive IDLapoE0/E0. There is growing evidence that binding of apoE4/E4 and apoE0/E0 to the flow sensor proteoheparan sulfate leads to nanoplaque build up [2] at the endothelium - blood interface and thus impairs vascular reactivity.

Insights Into Echo-Attenuated Plaques, Echolucent Plaques, and Plaques With Spotty Calcification: Novel Findings From Comparisons Among Intravascular Ultrasound, Near-Infrared Spectroscopy, and Pathological Histology in 2,294 Human Coronary Artery Segments

Three intravascular ultrasound (IVUS) signatures have been associated with coronary artery disease instability: echo attenuation, an intraplaque echolucent zone, and spotty calcification. The aim of this study was to investigate the substrates responsible for these IVUS signatures in a relatively large series of post-mortem human coronary samples. The exact mechanisms and pathological correlates underlying echo attenuation, an intraplaque echolucent zone, and spotty calcification remain poorly understood. IVUS was compared with near-infrared spectroscopic detection of lipid core plaque and histopathology in 2,294 vessel segments from 151 coronary specimens from 62 patients at necropsy using the modified American Heart Association classification. IVUS detected echo-attenuated plaques in 18.3% of segments, echolucent plaques in 10.5% of segments, and spotty calcification in 14.4% of segments. Histopathologically, 91.4% of echo-attenuated plaques corresponded to either a fibroatheroma (FA) with a necrotic core (NC) or pathological intimal thickening with a lipid pool; almost all segments with superficial echo attenuation indicated the presence of an FA with an advanced NC. Echolucent plaques indicated the presence of a relatively smaller lipid or NC compared with echo-attenuated plaques (thickness: 0.51 mm [interquartile range (IQR): 0.35 to 0.64 mm] vs. 0.70 mm [IQR: 0.54 to 0.92 mm] [p < 0.001]; arc: 74.5° [IQR: 59.0° to 101.0°] vs. 90° [IQR: 70.0° to 112.0°] [p < 0.001]), although 82.8% of superficial echolucent zones indicated an NC-containing FA. IVUS spotty calcification, especially when superficial in location (72.6%), was often associated with an FA with calcium deposits and had smaller arcs of calcium in the setting of FA compared with fibrocalcific plaques (37.5° [IQR: 23.0° to 53.0°] vs. 59.0° [IQR: 46.0° to 69.0°]; p < 0.001). Comparisons between IVUS and near-infrared spectroscopy revealed that echo-attenuated plaques contained the highest probability of near-infrared spectroscopy-derived lipid core plaque, followed by echolucent plaques and spotty calcifications. This study demonstrated that echo-attenuated plaque, especially superficial echo attenuation, was the most reliable IVUS signature for identifying a high-risk plaque (i.e., an FA containing a large NC).

Molecular Imaging of Vascular Endothelial Growth Factor Receptors in Graft Arteriosclerosis

Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis. Graft arteriosclerosis is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors and vascular remodeling in human coronary artery grafts by molecular imaging. VEGF receptor specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells led to significant neointima formation in the grafts over a period of 4 weeks. Near-infrared fluorescent imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homolog of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGF receptor-1 but not VEGF receptor-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area. Molecular imaging of VEGF receptors may provide a noninvasive tool for detection of graft arteriosclerosis in solid organ transplantation.

Abstract 108: LDL-Apolipoprotein E Isoform Codetermines Human Coronary Artery Tone

Background: Apolipoprotein E4 (apoE4) is the most significant risk factor known to cause Alzheimer dementia (AD) [1], but also crucial in atherogenesis as pathological correlative of coronary heart disease (CHD) [2]. Therefore, the present investigation aimed at unveiling the role of LDL-apoE isoforms in vascular reactivity, especially in flow-dilatation of human coronaries. Materials and Methods: Isometric tension was measured in segments of human coronary arteries from heart transplantations changing the superfusate flow rate in the sequence 3, 5, 20, 40 and 100 mL/min. The initial tension was 2 g. Krebs solution without (control) and with addition of 100 mg/dL pooled LDL, LDLapoE4/E4 or LDLapoE0/E0 (from an apoE ‘knockout man’) was used as superfusate. Intracellular recordings of membrane potential and determination of cAMP and cGMP concentrations were carried out. Results: Initial tension at 3 mL/min flow (T 3 ): control 1.649 g, pooled LDL 1.739 g, LDLapoE4/E4 1.773 g, LDLapoE0/E0 1.796 g; flow-dependent Δtension (T 100 - T 3 ): control -0.418 g, pooled LDL -0.263 g, LDLapoE4/E4 -0.197 g, LDLapoE0/E0 +0.313 g. Upon a basal flow rate of 3 mL/min, the initial tension showed a graded course both in normal and in deendothelialized preparations: both pooled LDL (+5.5% vs. +4.7%) and LDLapoE4/E4 (+7.5% vs. +5.6%) as well as LDLapoE0/E0 (+8.9% vs. +10.2%) enhanced mechanical force as compared to the controls. Endothelium-denuded segments had basically a higher tone (+5.2%) on grounds of an impaired basal NO release. Latter preparations demonstrated this graded course especially significant during flow-dependent dilatation, which was reversed to constriction by +109.3% under pooled LDL, by +160.1% under LDLapoE4/E4 and by +332.1% under LDLapoE0/E0 compared with the control. Membrane potentials reflected tension: hyperpolarization of the vascular smooth muscle cells preceded relaxation, depolarization preceded contraction. Relating the membrane potential to the corresponding tone revealed a sigmoidal electromechanical coupling curve. For the first time in literature, we report on a chemomechanical coupling curve by correlating the nucleotide concentration with the corresponding tension. Conclusion: LDLapoE4/E4 reversed flow-dependent vasodilatation to constriction and was only exceeded in its deleterious effect by LDL devoid of any apoE. There is growing evidence that binding of apoE4/E4 and apoE0/E0 to the flow sensor proteoheparan sulfate (endothelial syndecan superfamily) leads to nanoplaque build-up [1,2] at the endothelium-blood interface and thus impairs vascular reactivity.

Neutralizing IL-6 Reduces Human Arterial Allograft Rejection by Allowing Emergence of CD161+ CD4+ Regulatory T Cells

Perioperative injuries to an allograft exacerbate graft rejection, which in humans is primarily mediated by effector memory T cells. IL-6 transcripts in human coronary artery segments rapidly increase posttransplantation into immunodeficient mouse hosts compared with those of pretransplant specimens and fall dramatically by 30 d. Adoptive transfer of human PBMCs allogeneic to the artery 2 d postoperatively results in T cell infiltrates and intimal expansion 4 wk later. Ab neutralization of human IL-6 reduces the magnitude of intimal expansion and total T cell infiltration but increases the relative expression of CD161 while decreasing other Th17 markers. Coculture of MHC class II-expressing human endothelial cells (ECs) with allogeneic CD4(+) memory T cells results in T cell activation and EC secretion of IL-6. Neutralizing IL-6 in primary allogeneic T cell-EC cocultures results in enhanced T cell proliferation of CD161(+) CD4(+) T cells, reduces total T cell proliferation upon restimulation in secondary cultures (an effect dependent on CD161(+) T cells), increases expression of FOXP3 in CD161(+) T cells, and generates T cells that suppress proliferation of freshly isolated T cells. These data suggest that IL-6 released from injured allograft vessels enhances allogeneic T cell infiltration and intimal expansion in a model of human allograft rejection by inhibiting an increase in CD161(+) regulatory T cells.

Biochemical characterization of atherosclerotic plaques by endogenous multispectral fluorescence lifetime imaging microscopy

Objective To investigate the potential of endogenous multispectral fluorescence lifetime imaging microscopy (FLIM) for biochemical characterization of human coronary atherosclerotic plaques. Methods Endogenous multispectral FLIM imaging was performed on the lumen of 58 segments of postmortem human coronary artery. The fluorescence was separated into three emission bands targeting the three main arterial endogenous fluorophores (390 ± 20 nm for collagen, 452 ± 22.5 nm for elastin, and 550 ± 20 for lipids). The fluorescence normalized intensity and average lifetime from each emission band was used to classify each pixel of an image as either “High-Collagen”, “High-Lipids” or “Low-Collagen/Lipids” via multiclass Fisher's linear discriminant analysis. Results Classification of plaques as either “High-Collagen”, “High-Lipids” or “Low-Collagen/Lipids” based on the endogenous multispectral FLIM was achieved with a sensitivity/specificity of 96/98%, 89/99%, and 99/99%, respectively, where histopathology served as the gold standard. Conclusion The endogenous multispectral FLIM approach we have taken, which can readily be adapted for in vivo intravascular catheter based imaging, is capable of reliably identifying plaques with high content of either collagen or lipids.

Influence of Ageing on Vasomotor Responses of Human Epicardial Coronary Arteries

Vasomotor responses to various agonists were studied on isolated circular segments of human epicardial coronary arteries from three different age groups; 23–38 years, 40–58 years and 63–86 years. Noradrenaline had no or only weak contractile effect on coronary arteries from younger patients but induced contraction of all artery segments from older patients. The Emax value was significantly (P<0.0001) higher in arteries from the oldest group compared to each of the two younger age groups, whereas the potency was similar in all three groups. Linear regression analysis of noradrenaline-induced contraction in individual patients revealed a significantly positive age-correlation (correlation coefficient 0.67, P<0.0001). Contraction induced by endothelin-1 and relaxation induced by substance P, calcitonin gene-related peptide and vasoactive intestinal peptide on arteries precontracted with U46619 showed no significant differences in maximum responses and potencies between the three age groups, and no significant linear age-correlation. These data demonstrate a large variability in contractile responses to noradrenaline with contractions seen mostly in coronary arteries from older patients. It thus seems that sympathetic activation could contribute to coronary ischaemia in some patients.

Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 μM), the neurokinin NK1 receptor antagonist L-733060 (0.5 μM), the voltage-sensitive calcium channel blocker ruthenium red (100 μM), the TRPV1 receptor antagonist capsazepine (5 μM), the nitric oxide synthetase inhibitor Nω-nitro-l-arginine methyl ester HCl (l-NAME; 100 μM), the gap junction blocker 18α-glycyrrhetinic acid (10 μM), as well as the RhoA kinase inhibitor Y-27632 (1 μM). Further, we also used the K+ channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 μM) + apamin (0.1 μM) and iberiotoxin (0.5 μM) + apamin (0.1 μM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) α-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.

Cholesterol crystals rupture biological membranes and human plaques during acute cardiovascular events‐a novel insight into plaque rupture by scanning electron microscopy

Plaque rupture and/or erosion are considered the leading cause of cardiovascular events. To elucidate this process, we demonstrated that during cholesterol crystallization the occupied volume increases rapidly and sharp-tipped crystals cut through thin biological membranes in their path. The amount of cholesterol correlated directly with both peak level and rate of crystal growth (r = 0.98; r = 0.99; p < 0.01, respectively). These observations suggest that crystallization of cholesterol in atherosclerotic plaques can induce cap rupture and/or erosion. Observations by scanning electron microscopy confirmed similar findings of cholesterol crystals perforating the lumen surface in human coronary artery segments with ruptured plaque.

Characterisation of CGRP receptors in human and porcine isolated coronary arteries: Evidence for CGRP receptor heterogeneity

This study sets out to characterise calcitonin gene-related peptide (CGRP) receptors in human and porcine isolated proximal and distal coronary arteries using BIBN4096BS. Human (h)-αCGRP induced relaxations that were blocked by BIBN4096BS in all arteries studied. In contrast to the other vessels, the Schild plot slope in the human distal coronary artery segments (0.68 ± 0.07) was significantly less than unity and BIBN4096BS potently blocked these responses (p K b (10 nM): 9.29 ± 0.34, n = 5). In the same preparation, h-αCGRP 8-37 behaved as a weak antagonist of h-αCGRP-induced relaxations (p K b (3 μM): 6.28 ± 0.17, n = 4), with also a Schild plot slope smaller than unity. The linear agonists, [ethylamide-Cys 2,7]-h-αCGRP ([Cys(Et) 2,7]-h-αCGRP) and [acetimidomethyl-Cys 2,7]-h-αCGRP ([Cys(Acm) 2,7]-h-αCGRP), had a high potency (pEC 50: 8.21 ± 0.25 and 7.25 ± 0.14, respectively), suggesting the presence of CGRP 2 receptors, while the potent blockade by BIBN4096BS (p K b (10 nM): 10.13 ± 0.29 and 9.95 ± 0.11, respectively) points to the presence of CGRP 1 receptors. Using RT-PCR, mRNAs encoding for the essential components for functional CGRP 1 receptors were demonstrated in both human proximal and distal coronary artery. Further, h-αCGRP (100 nM) increased cAMP levels, and this was attenuated by BIBN4096BS (1 μM). The above results demonstrate the presence of CGRP 1 receptors in all coronary artery segments investigated, but the human distal coronary artery segments seem to have an additional population of CGRP receptors not complying with the currently classified CGRP 1 or CGRP 2 receptors.