Caroline Cheng

Atherosclerotic Lesion Size and Vulnerability Are Determined by Patterns of Fluid Shear Stress

Background— Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies. Methods and Results— We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, 1.38±0.68 versus 0.22±0.04); contained fewer smooth muscle cells (1.9±1.6% versus 26.3±9.7%), less collagen (15.3±1.0% versus 22.2±1.0%), and more lipids (15.8±0.9% versus 10.2±0.5%); and showed more outward vascular remodeling (214±19% versus 117±9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II–induced intraplaque hemorrhages occurred in the lowered shear stress regions only. Conclusions— Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions.

Shear stress–induced changes in atherosclerotic plaque composition are modulated by chemokines

We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-gamma-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.

Toll like receptor 4 in atherosclerosis and plaque destabilization

The immune system plays a pivotal role in initiation and progression of atherosclerosis. Monocytes and T-lymphocytes are the first cells to enter the damaged endothelium. Differentiation of monocytes into macrophages and ingestion of lipids by these macrophages turning them into foam cells is a crucial step in the development of a fatty streak, the first sign of atherosclerosis. In recent years there has been accumulating evidence for the involvement of Toll like receptor 4, a pattern recognition receptor of the innate immune system, in the pathogenesis of atherosclerosis. Different cell types present in the atherosclerotic plaque express TLR4 and several pro-atherogenic ligands have been shown to activate TLR4. The innate immune system and the TLR signaling cascade may play an important role not only in the pathogenesis of atherosclerosis, but also in plaque destabilization. In this review, we discuss the role of TLR4 in the pathogenesis of atherosclerosis and vulnerable plaque development.

Augmentation of Wall Shear Stress Inhibits Neointimal Hyperplasia After Stent Implantation Inhibition Through Reduction of Inflammation

Background—Low wall shear stress (WSS) increases neointimal hyperplasia (NH) in vein grafts and stents. We studied the causal relationship between WSS and NH formation in stents by locally increasing WSS with a flow divider (Anti-Restenotic Diffuser, Endoart SA) placed in the center of the stent. Methods and Results—In 9 rabbits fed a high-cholesterol diet for 2 months to induce endothelial dysfunction, 18 stents were implanted in the right and left external iliac arteries (1 stent per vessel). Lumen diameters were measured by quantitative angiography before and after implantation and at 4-week follow-up, at which time, macrophage accumulation and interruption of the internal elastic lamina was determined. Cross sections of stent segments within the ARED (S+ARED), outside the ARED (S[minus]ARED), and in corresponding segments of the contralateral control stent (SCTRL) were analyzed. Changes in WSS induced by the ARED placement were derived by computational fluid dynamics. Computational fluid dynamics analysis demonstrated that WSS increased from 0.38 to 0.82 N/m2 in the S+ARED immediately after ARED placement. This augmentation of shear stress was accompanied by (1) lower mean late luminal loss by quantitative angiography ([minus]0.23±0.22 versus [minus]0.58±0.30 mm, P =0.02), (2) reduction in NH (1.48±0.58, 2.46±1.25, and 2.36±1.13 mm2, P <0.01, respectively, for S+ARED, S[minus]ARED, and SCTRL), and (3) a reduced inflammation score and a reduced injury score. Increments in shear stress did not change the relationship between injury score and NH or between inflammation score and NH. Conclusions—The newly developed ARED flow divider significantly increases WSS, and this local increment in WSS is accompanied by a local reduction in NH and a local reduction in inflammation and injury. The present study is therefore the first to provide direct evidence for an important modulating role of shear stress in in-stent neointimal hyperplasia.

Heme Oxygenase 1 Determines Atherosclerotic Lesion Progression Into a Vulnerable Plaque

Background— The molecular regulation for the transition from stable to vulnerable plaque remains to be elucidated. Heme oxygenase 1 (HO-1) and its metabolites have been implicated in the cytoprotective defense against oxidative injury in atherogenesis. In this study, we sought to assess the role of HO-1 in the progression toward plaque instability in carotid artery disease in patients and in a murine model of vulnerable plaque development. Methods and Results— Atherectomy biopsy from 112 patients with clinical carotid artery disease was collected and stratified according to characteristics of plaque vulnerability. HO-1 expression correlated closely with features of vulnerable human atheromatous plaque (P<0.005), including macrophage and lipid accumulation, and was inversely correlated with intraplaque vascular smooth muscle cells and collagen deposition. HO-1 expression levels correlated with the plaque destabilizing factors matrix metalloproteinase-9, interleukin-8, and interleukin-6. Likewise, in a vulnerable plaque model using apolipoprotein E−/− mice, HO-1 expression was upregulated in vulnerable versus stable lesions. HO-1 induction by cobalt protoporphyrin impeded lesion progression into vulnerable plaques, indicated by a reduction in necrotic core size and intraplaque lipid accumulation, whereas cap thickness and vascular smooth muscle cells were increased. In contrast, inhibition of HO-1 by zinc protoporphyrin augmented plaque vulnerability. Plaque stabilizing was prominent after adenoviral transduction of HO-1 compared with sham virus–treated animals, providing proof that the observed effects on plaque vulnerability were HO-1 specific. Conclusions— Here we demonstrate in a well-defined patient group and a murine vulnerable plaque model that HO-1 induction reverses plaque progression from a vulnerable plaque to a more stable phenotype as part of a compensatory atheroprotective response.

Connecting heart failure with preserved ejection fraction and renal dysfunction: the role of endothelial dysfunction and inflammation

Renal dysfunction in heart failure with preserved ejection fraction (HFpEF) is common and is associated with increased mortality. Impaired renal function is also a risk factor for developing HFpEF. A new paradigm for HFpEF, proposing a sequence of events leading to myocardial remodelling and dysfunction in HFpEF, was recently introduced, involving inflammatory, microvascular, and cardiac components. The kidney might play a key role in this systemic process. Renal impairment causes metabolic and systemic derangements in circulating factors, causing an activated systemic inflammatory state and endothelial dysfunction, which may lead to cardiomyocyte stiffening, hypertrophy, and interstitial fibrosis via cross‐talk between the endothelium and cardiomyocyte compartments. Here, we review the role of endothelial dysfunction and inflammation to explain the link between renal dysfunction and HFpEF, which allows for identification of new early risk markers, prognostic factors, and unique targets for intervention.

Nucleotide Excision DNA Repair Is Associated With Age-Related Vascular Dysfunction

Background Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. Methods and Results In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1d/− and XpdTTD mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1d/− mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1d/− mice, age-related endothelium-dependent vasodilator dysfunction in XpdTTD animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Conclusions Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

Capture of circulatory endothelial progenitor cells and accelerated re-endothelialization of a bio-engineered stent in human ex vivo shunt and rabbit denudation model

Aims The Genous™ Bio-engineered R™ stent (GS) aims to promote vascular healing by capture of circulatory endothelial progenitor cells (EPCs) to the surface of the stent struts, resulting in accelerated re-endothelialization. Here, we assessed the function of the GS in comparison to bare-metal stent (BMS), when exposed to the human and animal circulation. Methods and results First, 15 patients undergoing coronary angiography received an extracorporeal femoral arteriovenous (AV) shunt containing BMS and GS. Macroscopical mural thrombi were observed in BMS, whereas GS remained visibly clean. Confocal and scanning electron microscopic (SEM) analysis of GS showed an increase in strut coverage. Quantitative polymerase chain reaction (qPCR) analysis of captured cells on the GS demonstrated increased expression of endothelial markers KDR/VEGFR2 and E-selectin, and a decrease in pro-thrombogenic markers tissue factor pathway inhibitor and plasminogen activator inhibitor-1 compared with BMS. Secondly, a similar primate AV shunt model was used to validate these findings and occlusion of BMS was observed, while GS remained patent, as demonstrated by live imaging of indium-labelled platelets. Thirdly, in an in vitro cell-capture assay, GS struts showed increased coverage by EPCs, whereas monocyte coverage remained similar to BMS. Finally, the assessment of re-endothelialization was studied in a rabbit denudation model. Twenty animals received BMS and GS in the aorta and iliac arteries for 7 days. Scanning electron microscopic analysis showed a trend towards increased strut coverage, confirmed by qPCR analysis revealing increased levels of endothelial markers (Tie2, CD34, PCD31, and P-selectin) in GS. Conclusion In this proof-of-concept study, we have demonstrated that the bio-engineered EPC-capture stent, Genous™ R™ stent, is effective in EPC capture, resulting in accelerated re-endothelialization and reduced thrombogenicity.

Functional Expression of Endothelial Nitric Oxide Synthase Fused to Green Fluorescent Protein in Transgenic Mice

The activity of endothelial nitric oxide synthase (eNOS) is subject to complex transcriptional and post-translational regulation including the association with several proteins and variations in subcellular distribution. In the present study we describe a transgenic mouse model expressing eNOS fused to green fluorescent protein (GFP), which allows the study of localization and regulation of eNOS expression. We tested the functionality of eNOS in the eNOS-GFP mice. Expression of eNOS was restricted to the endothelial lining of blood vessels in various tissues tested, without appreciable expression in non-endothelial cells. Activity of the enzyme was confirmed by assaying the conversion of L-arginine to L-citrulline. NO production in isolated vessels was increased in transgenic mice when compared to non-transgenic control animals (4.88 +/- 0.59 and 2.48 +/- 0.47 micro mol/L NO, respectively, P < 0.005). Both the mean aortic pressure and the pulmonary artery pressure were reduced in eNOS-GFP mice (both approximately 30%, P < 0.05). Plasma cholesterol levels were also slightly reduced ( approximately 20%, P < 0.05). In conclusion, eNOS-GFP mice express functional eNOS and provide a unique model to study regulation of eNOS activity or eNOS-mediated vascular events, including response to ischemia, response to differences in shear stress, angiogenesis and vasculogenesis, and to study the subcellular distribution in relation with functional responses to these events.

Rheb Is Essential for Murine Development

ABSTRACT Ras homolog enriched in brain (Rheb) couples growth factor signaling to activation of the target of rapamycin complex 1 (TORC1). To study its role in mammals, we generated a Rheb knockout mouse. In contrast to mTOR or regulatory-associated protein of mTOR (Raptor) mutants, the inner cell mass of Rheb−/− embryos differentiated normally. Nevertheless, Rheb−/− embryos died around midgestation, most likely due to impaired development of the cardiovascular system. Rheb−/− embryonic fibroblasts showed decreased TORC1 activity, were smaller, and showed impaired proliferation. Rheb heterozygosity extended the life span of tuberous sclerosis complex 1-deficient (Tsc1−/−) embryos, indicating that there is a genetic interaction between the Tsc1 and Rheb genes in mouse.