Multiple Vascular Beds Research Articles

Atherosclerosis--multiple pathways to lesional macrophages.

Atherosclerosis is a chronic inflammatory disease that manifests in multiple vascular beds and frequently culminates in ischemic events, including myocardial infarction. Blood monocytes that are recruited to the inflamed vascular wall develop into inflammatory macrophages and foam cells, which contribute to pathogenesis at many stages of this disease and, therefore, represent a target for therapeutic interventions. Recently, alternate sources of macrophages have been identified. Here, we discuss the origin and molecular regulation of macrophages and highlight recent conceptual changes that may shape the development of effective treatments.

Abstract 333: Platelet and Endothelial Aggregation Receptor-1 Tempers Tubulogenesis via the Akt Pathway

Platelet endothelial aggregation receptor-1 (PEAR1) is a transmembrane receptor expressed on platelets and endothelial cells (ECs). We aim to unravel its function in ECs. qPCR analyses for PEAR1 in cultured human endothelial progenitors (blood outgrowth endothelial cells (BOECs)), in ECs from human umbilical cord (HUVECs and HUAECs) and in ECs freshly isolated from multiple vascular beds showed the lowest expression in BOECs and the highest in macro- and microvascular ECs from heart and liver. HUVECs and HUAECs showed intermediate PEAR1-expression. Staining of fast growing skin hemangiomas versus normal skin ECs revealed low expression of PEAR1 in rapidly proliferating tumor ECs. Short hairpin based miRNA lentiviral PEAR1-knockdown (shPEAR1) doubled the hybrid EA.hy926 and HUVEC EC proliferation rate compared to control lentiviral-transduced ECs. This was associated with downregulation of HES1-expression (a downstream regulator of the NOTCH-pathway), with increased baseline Akt-P levels (PI3K-activity) in shPEAR1 cells and with decreased levels of the phosphatase PTEN (PI3K-antagonist). Expression of the nuclear proliferation suppressor p21CIP1 in shPEAR1 ECs was reduced by 60±2% (mean±SEM, qPCR and Western blot), whereas the band intensity for p34/CDC2 significantly rose on Western blots (increased mitosis). PEAR1-knockdown also enhanced EC migration via reduced expression of β1- and β3-integrins both in HUVECs and EAhy926 ECs. Both enhanced proliferation and migration contributed to increased tube formation in shPEAR1 HUAECs (in vitro matrigel tube formation assays). During HUAEC tube formation, PEAR1-expression rose 5-fold, associated with a 3-fold increase of PTEN. However, PTEN-levels dropped 1,5-fold during tube formation of shPEAR1 HUAECs, compatible with increased phosphorylation levels of Akt in shPEAR1 ECs. Our study provides the first evidence that PEAR1-expression is variable in ECs from multiple vascular beds and that PEAR1 plays a suppressive role on proliferation, migration and tube formation of ECs through regulation of the PI3K/Akt/PTEN-pathway. Our data suggest a regulatory role for PEAR1 in the NOTCH-pathway via HES1. The upregulation of PEAR1 during EC maturation is compatible with this suppressive role.

Heart failure patients have impaired thermoregulatory response during exercise (884.2)

Objective: During a thermoregulatory challenge such as exercise in the heat, skin blood flow (SkBF) must rise so as to maximise radiant, convective and evaporative heat loss. There is some evidence to suggest that in Heart Failure (HF) the rise in SkBF is blunted during passive heat exposure. Indeed, HF patients may be unable to adequately increase cardiac output to service multiple vascular beds during a thermoregulatory challenge. The aim of this study was to compare the thermoregulatory responses to exercise in the heat in a group of HF patients (n=7, 62 + 7 yr, NYHA Class I‐II) and age‐matched healthy controls (CON; n=6, 63 + 8 yr). Methods: HF and CON exercised for 60 min heat (30oC, 60% relative humidity) at ~70% peak oxygen uptake (VO2). Core temperature (Tc) and skin temperature (Tsk) were measured continuously. Skin blood flow was measured every 10 minutes using Laser Doppler Flowmetry. Skin conductance was determined from the ratio of SkBF and mean arterial pressure. Results: HF patients exercised with a significantly (p<0.05) lower metabolic heat load when compared to CON (VO2: HF 1.26 + 0.32; CON 1.83 + 0.52 l/min). Despite this, both HF and CON had a similar and significant increase (P<0.05) in Tc (HF: pre: 37.2+ 0.4, end ex: 38.2 + 0.4oC; CON: pre 37.3 + 0.2, end ex: 38.2 + 0.4oC) and Tsk (HF: pre: 33.0+ 0.6, end ex: 35.0 + 0.5oC; CON: pre 32.6 + 0.6, end ex: 35.7 + 0.6oC). HF patients had a significantly lower (p<0.01) increase in SkBF (HF: 101 + 30; CON: 302 + 64 AU) and skin conductance (HF: 1.0 + 0.6; CON: 2.9 + 1.1 AU). Conclusion: Despite exercising at a lower metabolic heat load, HF patients had a similar increase in Tc but lower SkBF when compared CON. Our results suggest HF patients may have impaired thermoregulation secondary to blunted circulatory heat dissipation in HF.

Abstract P228: Distribution of Multisite Atherosclerosis in Persons with Metabolic Syndrome & Diabetes: The Multi-Ethnic Study of Atherosclerosis

Background: The extent of atherosclerosis across multiple vascular beds in persons with metabolic syndrome (Mets) and diabetes (DM) has not been documented. We hypothesized a greater presence/extent of multisite atherosclerosis in MetS and DM, vs. neither. Methods: We analyzed four vascular beds (coronary (CA), abdominal aorta (AA), carotid, peripheral vascular) in subjects without cardiovascular disease from the Multi-Ethnic Study of Atherosclerosis, who had complete data on these measures. Multisite atherosclerosis was defined as the presence/extent of CA calcium, AA calcium, carotid intimal medial thickness (disease ≥1mm) and borderline/abnormal (<1 or ≥1.3) ankle brachial index. We then created a composite score summing the continuous gender-stratified standardized z-scores for each measure. ANOVA compared least squares means across disease states; logistic regression gave odds of being in the top quartile of the composite score distribution. We adjusted for non-MetS risk factors (age, gender, ethnicity, smoking, LDL-C, lipid medications, family history). Results: Of 1,771 subjects (mean age 64, 50% male, 40% Caucasian, 25% Hispanic, 21% African American, 14% Chinese), 25% had MetS and 13% DM. Persons with DM or Mets had a greater number of sites positive for atherosclerosis ( figure ). Adjusted mean composite scores increased across disease groups (neither disease 0.24, MetS 0.88, DM 1.38, p<0.001 between groups). Adjusted logistic regression showed that the odds of being in the top quartile of the composite score were higher for those with DM OR=4.42 [2.21-8.83] p<0.001 and MetS OR=2.72 [1.63-4.56] p<0.001, vs. neither condition. Conclusions: Those with MetS or DM have an increased prevalence and extent of multisite atherosclerosis. Whether disease in one vascular bed should prompt further evaluation, and how this relates to future CVD events, requires further investigation. Our findings support the concept that atherosclerosis in those with MetS or DM is more likely to be systemic vs. site-specific.

Effect of calcitonin gene-related peptide antagonist on the mortality by nitrate induced tolerance rats with acute myocardial infarction

Calcitonin gene-related peptide (CGRP) is one of the most potent vasodilator, which dilates multiple vascular beds, but the coronary circulation is particularly sensitive. The purpose of this study was to evaluate the influence of CGRP antagonist on: 1. Acute mortality; 2. Ventricular arrhythmias and; 3. Prostaglandin E2 (PGE2) production by nitrate induced tolerance rats with myocardial infarction (MI).

Phenotypic, Genotypic, and Functional Characterization Of AML-Derived Endothelial Cells

Abstract Background In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to regulation of hematopoiesis with effects on cell proliferation and survival. Characteristics of marrow—derived endothelial cells from normal subjects have been described (Blood 1994; 84: 10-19), but characterization of endothelial cells in leukemia states is incomplete. Angiogenesis is known to be increased in AML marrows, and circulating endothelial progenitors are increased and correlate with disease status and response to treatment. Furthermore, cytokines secreted by endothelial cells such as vascular endothelial growth factor (VEGF) have been found to serve as growth factors for leukemia, sometimes in a paracrine or autocrine fashion. Despite these findings, inhibition of VEGF with agents such as bevacizumab has not demonstrated clinical anti-leukemia activity. Since our group and others have shown that endothelial cells from multiple vascular beds (human umbilical vein endothelial cells—HUVECs), human microvascular endothelial cells derived from skin (HMEC-1 cell line), and normal subject—derived endothelial cells are able to prevent spontaneous or therapy-induced apoptosis in AML blasts, it is important to understand the phenotype and characteristics of endothelial cells isolated from AML patients to understand their functional roles and to see if they might have an angiogenic gene expression profile as has been described in multiple myeloma (Clin Cancer Res 2009 15:5369). Methods Endothelial cells were purified from marrow aspirates obtained with consent from normal subjects or from newly diagnosed AML patients. Cells were isolated using anti-CD105-PE (BD Bioscience) followed by anti-PE microbead selection (Miltenyi™) or after disruption of marrow spicules with subsequent selection for endothelial cells in endothelial cell selective medium (EGM-2, Lonza). Cells between 2nd and 4th passage were utilized for analysis. Protein expression was determined by flow cytometry, Western blotting, or RT-PCR. Matrigel™ tubule formation and acetyl-LDL expression were determined as per previously published methods, as were adhesion, CFU-L, and transmigration assays. RNASeq was performed by the Functional Genomics Core at the University of Rochester after extraction of polyadenylated RNA from purified total RNA. Conversion to cDNA occurred with the Illumina TruSeq™ preparation kit, and sequencing was accomplished with the Illumina Genome Analyzer IIx. CASAVA software was utilized for analysis. Results Marrow derived endothelial cells from normal and AML subjects express CD105 (endoglin), CD31(PECAM), CD106 (VCAM), CD146 (MCAM), CD54 (ICAM), and CD34. They do not express CD14 nor CD45, and they demonstrate low level expression of CD144 (VE-cadherin). By RT-PCR, they express Tie-2, VEGF, and eNOS (endothelial nitric oxide synthase). They express acetyl-LDL and form tubular structures in Matrigel™. Phosphorylated components of the mTOR and PI3K/Akt pathways were also expressed by Western blot analysis. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and CFU-L outgrowth, but no significant differences were noted in these functions between normal and AML—derived endothelial cells in vitro assays. RNASeq analysis revealed 130 genes significantly up—or down—regulated in AML derived endothelial cells as compared with those derived from normal marrow. Endothelial cells from both sources had a distinct signature from marrow—derived fibroblasts. The genes differentially expressed (p<0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos, and this upregulation in AML vs. normal subject derived endothelial cells was confirmed by Western blot analysis. Genes involved in chemotaxis such as CXCL16 were also upregulated. Conclusions AML—derived endothelial cells exhibit similar phenotype and function as their normal marrow—derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Coagulation Factor VIII Is Synthesized In Endothelial Cells

Combined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive bleeding disorder resulting from mutations in Lman1. This gene encodes a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of factor V (FV) and factor VIII (FVIII) to the plasma. F5F8D is characterized by levels of both FV and FVIII reduced to ∼5-30% of normal. In contrast, Lman1 knockout mouse models of F5F8D exhibit FV and FVIII activities that are ∼50% of normal, relative to wildtype mice. Though FV and FVIII are synthesized at markedly different levels and potentially in different tissues, loss of the LMAN1 cargo receptor leads to parallel reductions in both FV and FVIII activity. FV is synthesized in hepatocytes (as well as megakaryocytes in the mouse). However, the primary cellular source of FVIII biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. We took advantage of the dependence of efficient FV and FVIII secretion on LMAN1 to examine the cellular source of each protein. FV and FVIII secretion profiles of conditional Lman1 knockout mice were characterized, relative to that of wildtype mice and ubiquitous Lman1 null mice (Lman1-/-). In order to generate mice with Lman1 expression specifically deleted in the endothelium or the hepatocytes, either a Tie2-Cre or Albumin-Cre transgene was crossed into Lman1 conditional mice (Lman1fl). FV and FVIII activity levels were measured by functional coagulation activity assays. Though Lman1fl/fl/Tie2-Cre+ mice (endothelial-specific knockout) exhibit normal plasma FV activity (99.9%) relative to wildtype mice (set to 100%), FVIII activity is reduced to 53.5% (p < 2.5 x 10-6). In contrast, Lman1fl/fl/Alb-Cre+ (hepatocyte-specific knockout) mice demonstrate normal FVIII activity (89.0%) and reduced FV activity (37.0%) (p < 1.4 x 10-10). To confirm endothelial cells as the biosynthetic source of FVIII, we took advantage of the previously reported RiboTag mouse (Sanz et al., 2009. PNAS 106(33):13939-44) to isolate endothelial cell RNA for qPCR analysis from various murine tissues. RiboTag mice carry a hemaglutinin-tagged ribosomal protein that can be used for cell-type specific immunoprecipitation of polyribosomes and subsequent RNA analysis when crossed with a Cre-recombinase expressing animal. qPCR analyses of endothelial cell RNA isolated from total liver lysates of five RiboTag/Tie2-Cre+ mice demonstrated 10-20 fold enrichment for gene transcripts that are known to be endothelial-specific, such as Cdhs (12.1 fold enrichment, p < 8.0 x 10-3), Vcam1 (13.4 fold enrichment, p <1.1 x 10-5), and Vwf (15.3 fold enrichment, p < 7.0 x 10-4), as well as for FVIII transcripts (11.4 fold enrichment, p < 4.0 x 10-5). In contrast, this analysis demonstrated a statistically significant depletion (5-10 fold) of transcripts from many known hepatocyte-specific genes, including multiple coagulation factor genes. Similar examination of kidney endothelial cell RNA also demonstrated enrichment for FVIII transcripts, thereby demonstrating that endothelial cells from multiple tissues and vascular beds contribute to the plasma FVIII pool in the mouse. These results explain the successful reversal of hemophilia A by both liver and kidney transplants. Taken together, these results definitively demonstrate that endothelial cells are the primary source of FVIII biosynthesis in the mouse, and that hepatocytes make no significant contribution to the plasma FVIII pool. Disclosures:No relevant conflicts of interest to declare.

Cone beam CT incidental findings: intracranial carotid artery calcification—a cause for concern

You may wonder why, as an exception, this editorial is co-authored by Arthur Friedlander from UCLA Dental School, LA. Well, he is currently actively involved in a discussion of obvious importance to the DMFR community. A recently published article noted that intracranial carotid artery calcifications (ICACs) were identified in 4 (2.4%) individuals (statistics recalculated by this correspondent) who were likely nested in the oldest cohort (n=169; age range, 50–85 years) of dental school clinic attendees having cone beam CT scans, in the main for implant placement.1 Using the best available evidence at the time, the authors concluded that no referral or intervention was warranted for these individuals because a study involving 406 (mainly Caucasian) patients (males, 60%; mean age, 62 ± 14 years) with ischaemic cerebrovascular symptoms (amaurosis fugax, transient ischaemic attack, stroke) could not associate the presence or volume of ICAC (as determined by multidetector CT angiography) with the patients’ symptomatic side.2 A review of more recent research, however, demonstrates the need to refer such patients to a physician because the data demonstrate that ICAC is a risk indicator of atherosclerotic disease burden across ethnic lines in both the cerebrovascular and cardiovascular distribution of healthy and symptomatic individuals. Specifically, the results of a recent study completed in Rotterdam, Netherlands, demonstrated that ICAC evidenced on non-contrast enhanced CT was prominently associated with the concomitant presence of MRI markers of vascular brain disease in healthy individuals.3 This investigation, conducted amongst 885 community-dwelling adults (males, 49.2%; mean age, 66.7 ± 5.5 years), demonstrated that higher calcification load and volume in the ICAC were prominently associated with MRI-determined increasing volume of white matter lesions known to be associated over time with development of cognitive impairment. These findings are substantiated by a study conducted in the USA which evaluated 108 consecutive multiethnic (African-American, Hispanic, Caucasian) patients (males 46%; mean age, 57.3 ± 12 years) admitted to the stroke service at Parkland Hospital in Dallas, TX.4 The researchers noted that extensive calcification of the ICAC on head CT was associated with high-grade stenosis (greater than or equal to 50%) on follow-up catheter-based cerebral angiographic study. These findings are supported by two other studies conducted in the Republic of Korea among groups of Asian patients admitted to major medical centers with symptomatic (acute ischaemic stroke/transient ischaemic attack) cerebrovascular disease. In the first project conducted amongst 159 consecutively treated patients (males, 61%; mean age, 66 ± 12 years), the extent of ICAC calcifications (as determined by unenhanced CT) was significantly associated with MRI-demonstrated periventricular and deep white matter hyperintensities and lacunar lesions.5 In the second Korean study evaluated, 314 patients (males, 65%; mean age, 66.3 ± 12.6 years), the severity of ICAC calcification on brain CT was significantly correlated with coronary artery calcium scores as determined by CT coronary angiography among patients previously believed free of atherosclerotic heart disease.6 Atherosclerosis is a generalized process affecting multiple vascular beds with arterial calcifications associated with more advanced states of disease. Cerebral calcification is a validated risk indicator of a diffuse atherosclerotic process in cerebral circulation and coronary artery atherosclerosis. The intention of this editorial is to make oral and maxillofacial radiologists aware that ICAC is a severe incidental finding that should definitely result in referral of these patients to physicians for further evaluation.

Platelet endothelial aggregation receptor-1 is a critical determinant of endothelial cell function

Background: Platelet endothelial aggregation receptor-1 (PEAR1), a type I transmembrane receptor, is expressed on platelets and endothelial cells (ECs) but its contribution to EC function is unknown. We aim to unravel its (patho)physiological role in ECs. Methods and results: PEAR1 expression was analyzed in cultured human endothelial progenitors (blood outgrowth endothelial cells or BOECs), in ECs from human umbilical cord and in ECs freshly isolated from multiple vascular beds (qPCR). mRNA expression was heterogeneous, with the lowest expression in BOECs and the highest in macrovascular and microvascular ECs from heart and liver. Umbilical cord ECs showed intermediate PEAR1 levels. Stainings of human hemangiomas (pyogenic granuloma) revealed low expression of PEAR1 in the young, rapidly proliferating ECs of the granuloma compared to normal subcutaneous ECs. The role of PEAR1 in EC proliferation was evaluated in the hybrid cell line EA.hy926. PEAR1 knockdown by lentiviral transduction (short-hairpin anti-PEAR1 construct; shPEAR1) doubled the EC proliferation rate, as compared to control lentiviral-transduced cells. This was associated with significantly increased baseline Akt-P levels (PI3K-activity) in shPEAR1 cells and significantly decreased levels of the phosphatase PTEN (PI3K-antagonist). Expression of the proliferation suppressor p21/CIP1 in shPEAR1 ECs was reduced by 60±2% (mean±SEM, qPCR), whereas the band intensity for CDC2 significantly rose on western blots, consistent with increased mitosis. In in vitro matrigel tube formation assays, using human umbilical artery endothelial cells (HUAECs), PEAR1 knockdown significantly (p<0.005) increased total tube length and number of tube branching points compared to control HUAEC tubes. PEAR1 expression rose 5-fold during tube formation. This was associated with a 3-fold increase of PTEN whereas PTEN-levels dropped 1,5-fold during tube formation of PEAR1-knockdown HUAECs, compatible with increased phosphorylation of Akt in shPEAR1 ECs. Anti-PEAR1 antibodies behaved as a pseudo-ligand in EA.hy926 ECs and HUAECs and triggered PEAR1- and Akt-phosphorylation. In turn, this resulted in PEAR1/Akt-mediated phosphorylation of eNOS (activating site P1177 on western blot) and in anti-PEAR1 pseudoligand stimulated tube formation. Conclusions: We provide the first evidence that PEAR1 is expressed in ECs from multiple vascular beds and that PEAR1 plays a suppressive role on proliferation, eNOS-activation and tube formation through the PI3K/Akt/PTEN pathway. The upregulation of PEAR1 during EC maturation is compatible with this suppressive role.

Contemporary Diagnosis of Carotid Fibromuscular Dysplasia: Role of Power Doppler and a Review of Other Diagnostic Modalities

Fibromuscular dysplasia (FMD) is a nonatheromatous, noninflammatory arteriopathy with segmental involvement of medium-sized arteries in multiple vascular beds. It most commonly involves the renal and carotid arteries. The etiology is unknown, although various hormonal and mechanical factors have been suggested. The disease can occur at any age but is usually diagnosed in middle-aged individuals, predominantly women. FMD is much more common than previously thought, perhaps affecting as many as 4% of adult women. Clinical manifestations of the internal carotid artery involvement are transitory ischemic attacks or cerebral infarction, as well as nonspecific symptoms such as headache and vertigo. In cases of cerebrovascular events, endovascular or surgical treatment is recommended; therefore, detection of FMD is of considerable importance. The gold standard for diagnosing FMD is catheter angiography (with the classic "string of beads" pattern), but this invasive procedure is only used for patients in whom it is clinically pertinent to proceed with revascularization. The optimal noninvasive modality for diagnosis and quantification for FMD is not known and little information has been recently published about new diagnostic modalities. Although angiography, computed tomography angiography, and magnetic resonance angiography are excellent in confirming the morphologic diagnosis of FMD, they are less accurate in assessing the hemodynamic significance of the lesions. Ultrasonography is useful in assessing the degree of carotid artery stenosis. Use of power Doppler ultrasound improves the ability to detect the morphologic features of carotid FMD.

Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

BackgroundArachidonic acid (AA) and/or its enzymatic metabolites are important lipid mediators contributing to endothelium‐derived hyperpolarizing factor (EDHF)–mediated dilation in multiple vascular beds, including human coronary arterioles (HCAs). However, the mechanisms of action of these lipid mediators in endothelial cells (ECs) remain incompletely defined. In this study, we investigated the role of the transient receptor potential vanilloid 4 (TRPV4) channel in AA‐induced endothelial Ca2+ response and dilation of HCAs.Methods and ResultsAA induced concentration‐dependent dilation in isolated HCAs. The dilation was largely abolished by the TRPV4 antagonist RN‐1734 and by inhibition of endothelial Ca2+‐activated K+ channels. In native and TRPV4‐overexpressing human coronary artery ECs (HCAECs), AA increased intracellular Ca2+ concentration ([Ca2+]i), which was mediated by TRPV4‐dependent Ca2+ entry. The AA‐induced [Ca2+]i increase was inhibited by cytochrome P450 (CYP) inhibitors. Surprisingly, the CYP metabolites of AA, epoxyeicosatrienoic acids (EETs), were much less potent activators of TRPV4, and CYP inhibitors did not affect EET production in HCAECs. Apart from its effect on [Ca2+]i, AA induced endothelial hyperpolarization, and this effect was required for Ca2+ entry through TRPV4. AA‐induced and TRPV4‐mediated Ca2+ entry was also inhibited by the protein kinase A inhibitor PKI. TRPV4 exhibited a basal level of phosphorylation, which was inhibited by PKI. Patch‐clamp studies indicated that AA activated TRPV4 single‐channel currents in cell‐attached and inside‐out patches of HCAECs.ConclusionsAA dilates HCAs through a novel mechanism involving endothelial TRPV4 channel‐dependent Ca2+ entry that requires endothelial hyperpolarization, PKA‐mediated basal phosphorylation of TRPV4, and direct activation of TRPV4 channels by AA.

Advances in Molecular Imaging: Plaque Imaging

Recent advances in nuclear plaque imaging aim to achieve noninvasive identification of vulnerable atherosclerotic plaques using positron emission tomography (PET) with 18F-fluorodexoyglucose (FDG) and novel tracers targeting molecular markers of inflammation and other active metabolic processes. Nuclear imaging of atherosclerosis has been demonstrated in multiple vascular beds, including the carotid, aorta, peripheral and coronary arteries—but significant challenges remain, especially for coronary imaging. The advantage of PET over other molecular imaging modalities is its superior sensitivity, however, low spatial resolution means that images must be co-registered with computed tomography (CT) or magnetic resonance imaging (MRI) for precise anatomical localization of the PET signal. Such hybrid techniques provide the best hope for early detection of prospective culprit lesions—which may, in the coronary vasculature, appear falsely low-risk using conventional coronary angiography or stress imaging. Current hot topics in nuclear plaque imaging include the use of FDG-PET for therapeutic monitoring in drug development, identification of imaging biomarkers to evaluate cardiovascular risk, and the development of novel tracers against an array of biologically important markers of atherosclerosis. The purpose of this article is to review these recent advances in nuclear plaque imaging.

A 67‐Year‐Old Woman Receiving Tumor Necrosis Factor α Inhibitor Therapy Presenting With Neurologic Dysfunction

A 67‐Year‐Old Woman Receiving Tumor Necrosis Factor α Inhibitor Therapy Presenting With Neurologic Dysfunction

GENETIC DESIGN OF PIGS AS EXPERIMENTAL MODELS IN THE COMBAT BETWEEN CHRONIC DISEASES AND HEALTHY AGING

Chronic, dysregulatory disease processes are becoming a major medical problem in the aging populations of our world. Metabolic syndrome (obesity, diabetes, atherosclerosis, and hypertension), cardiovascular diseases, neurodegeneration (e.g. Alzheimer syndrome), inflammatory diseases, and cancers affect a rapidly increasing number of people. The sequencing of the human genome should help in disease prevention by allowing mapping and characterisation of “illness” and “wellness” gene variants that convey susceptibility or resistance to the dysregulatory and degenerative disease processes. Common chronic disorders are genetically very heterogeneous with some subtypes showing Mendelian inheritance with quite high penetrance. Finding such monogenetic causes will allow truly personalized prevention and treatment. However, genetics is just the first step – functional studies in model systems will be necessary. The pig is an excellent model for medical research as well as for testing of new methods and drugs for disease prevention and treatment. Its size and longevity makes it especially useful for the study of chronic disease processes that can be monitored and repeatedly biopsied for long periods with and without intervention. The genome of different pig breeds have been sequenced, revealing that the pig is genetically more similar to man than conventional laboratory animals – in agreement with the similarities in organ development, physiology and metabolism. Genetically designed minipigs (Göttingen and Yucatan) are obtained by genetic engineering of somatic cells and animal cloning by somatic cell nuclear transfer. Primary minipig fibroblasts are genetically modified in culture by transposon-based transgenesis and/or homologous recombination with AAV-transduced constructs. The designed pig cells are electro-fused with enucleated oocytes (from normal slaughtered pigs) and the reconstructed oocytes develop in vitro into blastocysts that are transferred to surrogate production sows giving birth to clones of the designed pigs. Our HMC (hand made cloning) technology is very cost-efficient and allows large-scale production, without a need for micromanipulation. Thus, minipigs have been produced that should be prone to develop disease processes such as neurodegeneration (dominant negative human AAP and/or PS1 expression), atherosclerosis (human gain-of-function PCSK9 expression, ApoE knockout, LDL-R knockout), inflammation (ectopic expression of human α2 and β1 integrins in suprabasal epidermis), and cancer (BRCA1 knockout). Interesting phenotypes are observed in many of these minipigs. The Yucatan pigs with liver-specific expression of gain-of-function PCSK9, for example, display reduced hepatic LDL-R levels, impaired LDL clearance, severe hypercholesterolemia with accumulation of ApoB100-containing lipoproteins, and spontaneous development of progressive atherosclerotic lesions in multiple vascular beds. The genetic load can be further increased or modulated by breeding or cross-breeding of the different model pigs. We can also produce clones of pigs, some disease prone and some fluorescing, to perform experiments in regenerative medicine where the fate of healthy fluorescent cells can be followed in the, basically identical, disease prone animals. It is also our hope that our pig models can contribute to the digital revolution in medicine, combining detailed genomic sequencing and analysis with the introduction of wireless biosensors and advanced imaging methods. “Digitalized” pigs should teach us how to apply these fantastic new possibilities clinically. We envisage that this will become one of the biggest shake-ups in the history of medicine.

Expression of pannexin isoforms in the murine arterial network

Pannexins form ATP‐release channels that have both physiological and pathological roles in cells such as regulation of vascular tone and apoptosis, respectively. However, distribution of pannexins across the vasculature is poorly documented. Therefore, we aimed to detail their distribution across different vascular beds. We first tested antibodies against pannexin 1 (Panx1), Panx2 and Panx3 on HEK cells (which do not endogenously express pannexins) transfected with plasmids for each pannexin isoform. Each of the pannexin antibodies was found to be specific. By comparing expression in C57Bl/6 and Panx1−/− mice, we demonstrate that in the systemic arterial network, Panx1 is the only isoform detected. In the large arteries and coronary circulation, Panx1 was primarily found in the endothelial cells, whereas in small arteries and arteioles Panx1 localizes to the smooth muscle cells. In the kidney, pulmonary and hepatic circulations, there was more variability in the pannexin isoforms detected. Pannexins were not detected at myoendothelial junctions across multiple vascular beds. We conclude that the specific localized expression of pannexin channels throughout the vasculature under physiological conditions points towards an important role for these channels in the regulation of ATP release throughout the arterial network.

Best surgical option for thoracoabdominal aneurysm repair - the open approach.

Open thoracoabdominal aneurysm (TAAA) repair is a complex surgical procedure that requires efficient replacement of diseased aorta, limitation of ischemia to the various and multiple vascular beds, and intraoperative protection of the heart, central nervous system and abdominal viscera in a patient population with multiple comorbidities. Techniques of TAAA repair have evolved from the original “clamp and sew” technique to modern perfusion-assisted techniques with varying degrees of hypothermia. In experienced aortic centers, the morbidity and mortality of TAAA repair is quite good with mortality 5.0% to 8.0%, paralysis 2.3% to 3.8%, and renal failure requiring hemodialysis 1.5% to 5.6%. Open repair is durable and the long-term survival is well-defined and acceptable. In our hands, the 10 year survival of open descending thoracic and thoracoabdominal aneurysm repair is 53.5%. The applicability of open TAAA repair to the myriad of aortic pathologies, anatomy and connective tissue disorders (CTDs) is unlimited. Thoracic endovascular aortic repair (TEVAR) is a less invasive method for repairing descending thoracic and thoracoabdominal aneurysms. Currently, the approved indication is repair of descending thoracic atherosclerotic aneurysms with adequate proximal and distal landing zones, although these devices are used to treat all aortic pathologies. The applicability of TEVAR to the transverse arch and visceral abdominal aorta has been extended using extraanatomical bypasses (hybrid techniques) or branched and/or fenestrated devices. Long-term durability of these repairs extended beyond the descending thoracic aorta is unknown. Furthermore, the application of TEVAR in patients with CTDs is not recommended in the STS guidelines for thoracic and thoracoabdominal aneurysm repair. As the next generation of devices become available (some with FDA indications other than atherosclerotic descending thoracic aneurysm) and as more data accumulates regarding the long-term efficacy of TEVAR, we may gain more insight into the specific clinical scenarios in which TEVAR may be superior to open repair. Currently, we believe that open repair in the hands of experienced surgeons is superior to TEVAR for thoracoabdominal aneurysms in a variety of aortic pathologies including: chronic aortic dissection, aortic aneurysm or dissection in patients with CTDs, mycotic pseudoaneurysm and infected aortic grafts. TEVAR has distinct advantages in other aortic pathologies, including acute complicated type B aortic dissection, blunt traumatic aortic injury (BTAI) and anatomically suitable ruptured descending thoracic aneurysm. Intimate knowledge of and facility in applying both techniques is paramount in deciding how to intervene in specific patient scenarios. The remainder of this commentary will discuss our opinion on open vs. endovascular repair of specific pathologies of the thoracoabdominal aorta.

Abstract P210: Calcified Atherosclerotic Plaque and All Cause Mortality in Diabetes: Diabetes Heart Study

Introduction Coronary artery calcium (CAC), a measure of atherosclerosis predicts mortality in both diabetes and the general population. The incremental utility of measuring atherosclerosis across multiple vascular beds beyond CAC for the prediction of mortality has not been reported. In this study we derived a composite atherosclerosis score (CAS) from multiple vascular beds and compared it with CAC in predicting all-cause mortality. We hypothesized that this composite score will be significantly better than CAC in predicting mortality. Methods A total of 803 participants, ages 39–86, with complete data on diabetes and vascular imaging in the Diabetes Heart Study (DHS) were followed for an average of 7.4 years. Computed tomography (CT) scans were performed at baseline to obtain measures of carotid (CAAC), coronary (CAC) and abdominal aorta (AAC) Agatston scores. A principal component analysis using studentized residuals of log transformed (CAAC+1), (CAC+1), and (AAC+1) adjusting for age, race, and gender was performed. We selected the first principal component as the CAS. Seven-year risk estimates for mortality were obtained using logistic regression models. Model 1 included age, gender, smoking, systolic blood pressure, antihypertensive medication use, total and high-density lipoprotein cholesterol, and ethnicity and CAS. Model 2 included these risk factors plus CAC. We compared the estimation of mortality in Model 1 with CAS vs. Model 2 with CAC using chi-square values. Results Overall, 14% (116/803) of participants died during follow-up. CAS explained 70% of the variance, (eigenvalue of 2.1 with loading, CAAC, 0.57; CAC, 0.57; and AAC, 0.59). After adjusting for potential confounders, the odds ratio (95% CI) of all-cause mortality for 1-standard deviation (SD) increment was 2.12 (1.64–2.78) for CAS and 2.38 (1.77–3.35) for CAC. The area under the curve (chi-square value) with CAS or CAC to predict mortality was 0.76 (36) vs 0.76 (37) respectively. Conclusion Subclinical atherosclerosis, as measured by CT determined calcified plaque burden has increasing evidence supporting its role as a tool to stratify future risk for mortality. Here we demonstrated that the diagnostic accuracy between CAS and CAC are comparable and the predictive value of CAC alone for mortality is not further enhanced by inclusion of calcified plaque burden in carotid or abdominal aortic territories.

The Association Between Abdominal Body Composition and Vascular Calcification

Subclinical cardiovascular disease (CVD) may be associated with both adipose and skeletal muscle tissues in the abdomen. Accordingly, we examined whether subcutaneous, intermuscular, and visceral adipose tissue, as well as abdominal lean muscle, were associated with the presence and extent of vascular calcification in multiple vascular beds. Three hundred and ninety four patients (58.1% men) underwent electron beam computed tomography (EBCT) scans as part of routine health maintenance screening. The coronary and carotid calcium scores were analyzed at the time of the scan, whereas the other calcium scores, as well as the body composition analyses, were analyzed retrospectively. Mean age was 55.2 ± 11.1 years and BMI was 26.9 ± 4.2. The prevalence of any calcification in the carotids, coronaries, thoracic aorta, abdominal aorta, and iliacs was 30.1, 60.1, 39.8, 55.7, and 56.8%, respectively. Compared to those with calcification in different vascular beds, those without vascular calcification generally had significantly more lean muscle and less adipose tissue. In separate multivariable logistic models, a 1 s.d. increment in the ratio of abdominal and visceral fat to total area of each corresponding compartments was significantly associated with an increased odds for the presence of thoracic aortic calcium (odds ratio (OR) = 1.6, 1.5, respectively; P = 0.01 for both). Conversely, increases in abdominal lean muscle were associated with significantly decreased odds of thoracic aortic calcification (OR = 0.34; P ≤ 0.01). A similar pattern of associations existed among the other vascular beds. Also, the association between lean muscle and vascular calcification was independent of visceral adipose tissue. In conclusion, adipose tissue was positively and lean body mass inversely associated with prevalent aortic calcification.

Increased expression of soluble fractalkine (CX3CL1) in systemic sclerosis – possible role in vascular inflammation

IntroductionThe pathogenesis of systemic sclerosis (SSc) involves interplay between obliterative vasculopathy in multiple vascular beds, inflammation, autoimmunity and progressive fibrosis. Vascular injury and activation are the earliest and possibly primary events in the pathogenesis of SSc. Aim of the workTo determine levels of serum soluble fractalkine (sFKN) and its receptor CX3CR1 in peripheral blood mononuclear cells (PBMCs) in systemic sclerosis (SSc) patients and healthy controls. In addition, to assess any possible association between sFKN and clinical features of SSc. Patients and methodsSerum levels of soluble fractalkine (sFKN, CX3CL1) assessed by enzyme linked immunosorbent assay (ELISA) and expression of its receptor (CX3CR1) on peripheral blood mononuclear cells by flow cytometric analysis, were measured in 18 systemic sclerosis (SSc) patients and 15 age and sex matched healthy controls. The degree of skin involvement was estimated by modified Rodnan skin thickness score (mRSS), pulmonary involvement was assessed in all patients by high resolution computerized tomography (HRCT) and pulmonary function tests (PFTs). ResultsSerum sFKN levels and expression of its receptor CX3CR1 were significantly elevated in SSc patients than in healthy controls (P<0.0.05). SSc patients with pulmonary fibrosis had sFKN levels three times higher than those without PF. Serum sFKN correlated inversely with forced vital capacity of lungs (FVC%) but correlated positively with severity of pulmonary fibrosis, extent of skin fibrosis (mRSS), pitting scars, skin ulcers, anti topo-isomerase1antibody and CRP. ConclusionSerum sFKN may play an important role in the pathogenesis of SSc, including tissue inflammation and vascular injury, hence, its measurement may be a useful serologic marker for the diagnosis and follow up of pulmonary and skin complications. So strategies to target CX3CL1–CX3CR1 interaction could provide a new therapeutic approach in SSc, potentially by blocking endothelial cell injury, leucocyte infiltration, and vascular injury.

Advances in Genetics 2010

The last year has seen several advances in genetics relevant to understanding the pathophysiology of stroke and to advancing diagnosis, prognosis, and care. ### Chromosome 9p21.3 Locus One of the most intriguing associations is that of a risk locus on chromosome 9p21.3 with ischemic stroke. This locus was first discovered by genomewide association to be a risk factor for coronary artery disease1,–,3 before studies showed a relationship with ischemic stroke.4,5 The locus also associates with intracranial and aortic aneurysms.6 The causative variant remains unknown. Recently, chromosome 9p21.3 has also been associated with platelet reactivity.7 This increased platelet reactivity may explain the association with myocardial infarction and stroke; it is less clear how increased platelet reactivity might relate to aneurysm formation. The 9p21.3 locus includes a noncoding RNA known as ANRIL, which in turn alters expression of several genes related to cellular proliferation.8 A richer explanation for the pleiotropic effects of the 9p21.3 locus on multiple vascular beds should emerge in the near future. ### Cerebral Autosomal-Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy and Conventional Risk Factors Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) remains incurable. However, recent clinical observations suggest that the …