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Extent of flow recirculation governs expression of atherosclerotic and thrombotic biomarkers in arterial bifurcations
Aims Atherogenesis, evolution of plaque, and outcomes following endovascular intervention depend heavily on the unique vascular architecture of each individual. Patient-specific, multiscale models able to correlate changes in microscopic cellular responses with relevant macroscopic flow, and structural conditions may help understand the progression of occlusive arterial disease, providing insights into how to mitigate adverse responses in specific settings and individuals.
Methods and results Vascular architectures mimicking coronary and carotid bifurcations were derived from clinical imaging and used to generate conjoint computational meshes for in silico analysis and biocompatible scaffolds for in vitro models. In parallel with three-dimensional flow simulations, geometrically realistic scaffolds were seeded with human smooth muscle cells (SMC) or endothelial cells and exposed to relevant, physiological flows. In vitro surrogates of endothelial health, atherosclerotic progression, and thrombosis were locally quantified and correlated best with an quantified extent of flow recirculation occurring within the bifurcation models. Oxidized low-density lipoprotein uptake, monocyte adhesion, and tissue factor expression locally rose up to three-fold, and phosphorylated endothelial nitric oxide synthase and Krüppel-like factor 2 decreased up to two-fold in recirculation areas. Isolated testing in straight-tube idealized constructs subject to static, oscillatory, and pulsatile conditions, indicative of different recirculant conditions corroborated these flow-mediated dependencies.
Conclusions Flow drives variations in vascular reactivity and vascular beds. Endothelial health was preserved by arterial flow but jeopardized in regions of flow recirculation in a quasi-linear manner. Similarly, SMC exposed to flow were more thrombogenic in large recirculating regions. Health, thrombosis, and atherosclerosis biomarkers correlate with the extent of recirculation in vascular cells lining certain vascular geometries.
- Flow
- Endothelium
- Geometry
- Atherosclerosis
- Thrombosis
1. Introduction
Endothelial cells (EC) are exposed to a complex biomechanical milieu and are responsible for relaying biochemical messages to underlying tissue. Disrupted flow patterns1,2 and, more specifically, flow recirculation exacerbate low-density lipoprotein internalization,3 cell adhesion molecules expression,4,5 and monocyte adhesion6,7 to the endothelium. Such events, in conjunction with other environmental factors,8 are early determinants for atherosclerosis progression.9 Though flow disruptions are evident in every vascular bifurcation, their magnitude depend heavily on the geometries specific to every patient and vessel.
The clinical treatment for atherosclerosis has tremendously evolved over many decades, with percutaneous interventions such as stents and/or grafts becoming almost a commodity in patient care.10 Device deployment, however, creates local, deep injury11 in the already fragile diseased vessel. Endothelial denudation12 exposes subintimal smooth muscle cells (SMC) to blood flow, and a cascade of reactions is elicited that modulates vessel patency and may trigger the extrinsic coagulation cascade.
Unravelling the molecular mechanisms behind vascular cell response to physiological and pathological flow characteristics has been for decades a matter of intense research.13 Both clinical data, obtained with tremendous advances in the field of bioimaging,8,14–16 and animal models have helped us understand how haemodynamics affect the vessel wall.17,18 Parallel in vitro work19–21 with idealized bench-top model systems has enabled the cellular and molecular examination of EC biological response to isolated flow descriptors (average flow, maximum amplitude, and flow frequency) in the presence and absence of SMC.
We now test the hypothesis that subtle variations in flow arising in different bifurcation settings are the most powerful predictors of biological markers crucial to our understanding of atherothrombotic disease. We examined how vessel-like, bench-top constructs derived from specific patient geometries create a more precise view of the underlying relationships between flow disruptions and local expressions of atherogenic and thrombotic markers. Using invasive22 and non-invasive imaging techniques,23 we reconstructed arterial geometries of different patients for use in a computational model, casted in vitro constructs based on the extracted geometry features, and seeded human vascular cells mimicking the arterial wall components. Computational fluid dynamic (CFD) simulations predicted physiological metrics of interest including velocity and quantified regions of flow recirculation. Bench-top-derived measurements of atherogenic and thrombotic markers and their geometry-specific variations were correlated with computational model-based predictions. A scalar metric, defined to capture the extent of recirculation for a specific geometry, correlated with oxidized low-density lipoprotein (Ox-LDL) uptake and localized monocyte adhesion to EC. Furthermore, SMC seeded in regions with a larger extent of recirculation increased their tissue factor (TF) expression. These observations point to the importance of accounting for patient-specific geometry variations and flow derangements to extend derived biological inferences beyond idealized cell culture models to real-world settings.
2. Methods
2.1 Arterial replication platform
Geometrical representations and flow wave forms of the left main coronary artery (LM) bifurcating into left anterior descending (LAD) and left circumflex (LCX) (Figure 1A and D), and the common carotid artery (CCA) bifurcating into the external (ECA) and internal (ICA) carotid arteries (Figure 1B and E), were needed as input values for in silico and in vitro experiments. Design of arterial mimics was performed using a modification of a previously developed computational framework24 (Figure 1C). EC (Figure 1F) and SMC (Figure 1G) were seeded on polydimethylsiloxane (PDMS) scaffolds. Scanning electron microscopy (SEM) revealed complete cellular coverage along length and circumference of the constructs (Figure 1H), and histological examination confirmed multilayer structure of SMC-coated constructs (Figure 1K). SMC (Figure 1I) and EC (Figure 1J) retained their cytoskeletal organization and alignment under flow. Vascular geometries and flow parameters, computational platform design, in silico models, and scaffold casting techniques are further detailed in Supplementary material online, Methods.
2.2 Recirculation metrics
The tangential component of the velocity vector (νTan), calculated as the dot product of local velocity vector and normalized direction vector representing the vessel centreline, was used to measure the velocity of the fluid flow (see Supplementary material online, Figure S2). This strategy is optimal in curved vessels since it always accounts for local recirculation. Based on νTan, extent of flow recirculation was classified into three categories: severe, mild, or negligible. If maximal backward velocity (νTanMIN) was above 20% of the inlet velocity during the pulse, extent of recirculation was considered severe. If νTanMIN was between 5 and 20%, extent was considered mild, and was negligible if the former criteria were not met. νTan was evaluated at each node and all tetrahedrons containing at least one node with negative νTan were summed to estimate the 'total recirculating volume' (VolRec) for each branch (Eq. 1). The parameter 'normalized νTan·VolRec' (NVV) was calculated multiplying VolRec by νTan, dividing it by the volume of the branch, and normalizing it to the value of the straight segment of the bifurcation (Eq. 2). NVV was the parameter found to best correlate disease biomarkers and flow disruptions (see Supplementary material online, Figure S3). $$mathtex$$\hbox{VolRec} = \sum {({\rm tetrahedron}{s_{\nu{\rm Tan \lt 0}}})}$$mathtex$$ (1) $$mathtex$$\hbox{NVV} = \displaystyle{{\nu \hbox{Tan} \cdot \hbox{VolRec}/\hbox{Vol Branch}} \over {(\nu\hbox{Tan} \cdot \hbox{VolRec}/\hbox{Vol Branch) Straight Branch}}}$$mathtex$$ (2)
2.3 Biological assessment of cell-seeded scaffolds
All biological results shown are extracted from the regions of interest. In straight-tube experiments, biomarkers of interest were analysed in the central region of the tube. In bifurcated experiments, data were extracted from the regions of recirculation indicated by CFD simulations (ECA, ICA, LAD, and LCX) and the centre of the straight upstream segments (CCA and LM). All results were normalized to their respective arterial flow (AF) values in single-channel experiments and to the straight sections in the bifurcations.
Vascular cell culture, seeding of scaffolds, perfusion bioreactor, Ox-LDL uptake, monocyte adhesion, vascular cell adhesion molecule 1 (VCAM-1), TF, phosphorylated endothelial nitric oxide synthase (p-eNOS) and Krüppel-like factor 2 (KLF-2) expression, KLF-2 mRNA expression, thrombogenicity, radioimmunoprecipitation assay extraction, western blot analysis, microscopic examination, ex vivo detection of macrophage presence in murine carotid bifurcations, and antibodies used are further detailed in Supplementary material online, Methods. All imaging analysis was done using the FIJI imaging platform.25
2.4 Human data and samples
All human data (angiographic images) were deidentified, and biological samples (EC, SMC, and blood) were from anonymous donors. Samples and data were treated following the guidelines of the Declaration of Helsinki.
2.5 Statistics
All in vitro experiments described were performed on triplicate specimens and repeated two separate times. In graphical presentations, data are expressed as average ± standard error of mean. Non-parametric Kruskal–Wallis test, followed by a Scheffé post hoc analysis of the original measured values normalized to their corresponding controls, was conducted to determine statistical differences between values. Values of P < 0.05 were considered statistically significant.
3. Results
3.1 Impact of flow patterns on markers of atherogenesis, and inflammation in EC, and thrombosis in SMC in straight constructs
In our idealized, straight constructs, we observed a protective effect on EC health under conditions of pulsatile unidirectional arterial flow in contrast to bidirectional oscillatory flow (OF) and static (STA) conditions. p-eNOS and KLF-2 expression were highest under AF, and were reduced significantly under OF or STA. KLF-2 mRNA expression was also muted in the oscillatory and static cases (data not shown) when compared with the arterial flow case.
VCAM-1 expression (Figure 2A), Ox-LDL uptake (Figure 2C), and cytokine-induced monocyte adhesion (Figure 2D) were highest under oscillatory or static conditions, and were reduced significantly under arterial flow. Activation by tumour necrosis factor alpha (TNF-α) amplified the signal in all cases. The normalized difference in expression of VCAM-1 was muted in activated EC (Figure 2B), but Ox-LDL uptake remained lower in the presence of arterial flow even in the face of cytokine stimulation. As expected, monocyte adhesion was below detection limits without TNF-α activation.
Similarly, arterial flow was protective of SMC lacking endothelial shielding. Basal TF levels rose 2.5-fold (Figure 2G and I–K) under oscillatory flow; in correlation, thrombus formation increased in our model to a similar extent (Figure 2H and L–N). TF expression by SMC was not affected by TNF-α activation. Thus, not only can disrupted flow exacerbate arterial injury but also the integrity of intact arterial flow may play an important protective role as well.
3.2 Predicting flow patterns in complex geometries
CFD calculations predicted recirculation downstream of bifurcation branches. Recirculation was observed in the two derivative branches of the carotid artery. Severe recirculation (νTanMIN = −195 mm/s) was observed in the ICA and mild recirculation (νTanMIN = −85 mm/s) in the ECA (Figure 3A). The extent of recirculation was four-fold higher in the ICA vs. the CCA (Figure 3C), while ECA's increase was around two-fold. An idealized model of the bifurcation of the LM was also studied. The two derivative branches, the LAD and the left circumflex (LCX) branches, presented recirculation that was mild, both in terms of flow reversal (Figure 3B) and extent of recirculation (Figure 3D). Simulation results were validated by tracking circulating injected latex microparticles.10
3.3 Expression of atherogenesis and thrombosis biomarkers correlates with the extent of flow recirculation in arterial bifurcations
Arterial flow appeared to protect EC from inflammatory factors in straight tubes seeded with vascular cells while oscillatory flow (as an idealized model of flow disruption) increased expression of cardiovascular disease biomarkers (Figure 2). The same array of tests was run in cell-seeded, 1 : 1 scale bifurcated models (Figures 4 and 5).
Phosphorylated eNOS (Figures 4A and 5A) and KLF-2 (Figures 4B and 5B) expression by EC were inversely correlated with NVV, with a 50% reduction for p-eNOS and up to 70% decrease for KLF-2. Contrarily, Ox-LDL uptake by EC (Figures 4D, 5D, see Supplementary material online, Figure S4A–C, and Table 1), monocyte adhesion to activated EC (Figures 4E, 5E, see Supplementary material online, Figure S4D–F, and Table 1), and TF expression in SMC (Figures 4F, 5F, see Supplementary material online, Figure S4G–I, and Table 1) correlated directly, and most powerfully, with the extent of recirculation in each branch of the studied bifurcations.
|Recirculation level/biomarker
|Negligible (AF, LM, and CCA)
|Mild (LAD, LCX, and ECA)
|Severe (ICA)
|Oscillatory flow
Ox-LDL uptake by EC increased with the intensity of recirculation, up to three-fold higher than upstream of the bifurcation divider (LM and CCA). Monocyte adhesion to activated EC also grew with the intensity of recirculation, doubling the recruitment by EC free of recirculation. VCAM-1 levels in activated EC, which were blunted in the straight-tube constructs, did not correlate with the observed flow regimes in our bifurcating models (Figures 4C, 5C, and Table 1) and were not detectable in the short sections analysed in the absence of TNF-α activation.
In SMC, TF levels were also sensitive to flow recirculation. SMC seeded in areas with severe recirculation showed a 2.5-fold increase in TF expression when compared with those seeded in regions exposed to negligible recirculation levels. The presence of macrophage was tested in carotid bifurcations of mice fed with a high-fat diet (see Supplementary material online, Figure S5). The extracted carotid geometry was simulated, and regions of flow stagnation near the vessel walls correlated fairly with macrophage recruitment.
4. Discussion
4.1 Flow patterns dictate EC and SMC reactivity
Vascular disease biomarkers and flow patterns are strongly correlated.5,27–29 In 1983, Zarins et al.27 observed that plaque formed predominantly in regions of flow separation and hypothesized that distinct wall shear stress profiles were responsible for such plaque localization. Dai et al.29 discovered how waveforms could be atheroprotective or atheroprone for EC, and could coexist simultaneously in different regions of the carotid bifurcation. In particular, cDNA array profiling proved that the two waveforms differentially stimulated genes involved in signal transduction, transcriptional regulation, inflammation, angiogenesis, coagulation, and lipid metabolism. They observed how atheroprotective waveforms enhanced KLF-2 and subsequently up-regulated eNOS, while atheroprone waveforms promoted up-regulation of interleukin-8, chemokine receptor type 4, and pentraxin 3 expression, which are biomarkers of atherosclerosis. Disturbed, atheroprone flow regimes usually comprise phases of reversal flow that generate oscillatory wall shear stresses, an attribute notably absent in atheroprotective flow regions.30 Oscillatory flow has been shown to up-regulate easily detectable cell adhesion molecules such as VCAM-1 or ICAM-1.31 We chose VCAM-1 as one of the atherogenesis biomarkers instead of ICAM-1, since VCAM-1 seems to be more critical at the beginning of lesion formation.32 We also chose Ox-LDL uptake and monocyte adhesion, which are fundamental players in atherosclerosis progression.33 Ox-LDL binds to scavenger receptors CD3634 and LOX-1,35 and ligation of LOX-1 induces NF-κB in EC.36 Transcriptional activation of genes encoding for LDL receptor, with the consequent enhancement of LDL uptake, are increased in EC in disturbed flow regions.37 Oscillatory flow increases native38 and Ox-LDL mediated monocyte chemotactic protein-1 expression in EC and subsequent monocyte adhesion.39 In the same study, high shear stress prevented both Ox-LDL uptake and monocyte adhesion. Depending on the degree of oxidation, LDL induces expression of P-selectin in human umbilical vein endothelial cells, which increases adhesion of monocytes.6
The literature is equivocal regarding the effects of haemodynamic forces on the coagulation cascade. While certain groups have reported an increase of TF expression,40 gene transcription and increased Factor Xa concentration41 in EC exposed to high shear stress, others affirmed that high shear stress provokes release of the TF pathway inhibitor from EC,42 reducing TF activity, and hence risk of thrombosis. Other authors reported that TF activity was up-regulated by oscillatory shear stress,43 and some noted that TF is of only modest importance in EC.44 While the effects of haemodynamic forces on TF in EC have been deeply explored, the influence of flow on SMC function is poorly characterized.45 Indeed, the endothelium is denuded after angioplasty46 and SMC are regularly exposed to blood flow. We therefore exposed single cultures of SMC to pulsatile flow and oscillatory flow along straight tubes and bifurcations, and analysed TF levels to elucidate risk of thrombosis as a function of flow. Although the increase of thrombogenicity may not only be caused by the increase of TF levels, in our system, TF expression in SMC correlated with ex vivo clot formation.
4.2 The extent of recirculation simplifies the relationship between geometry and flow and correlates with early markers of EC and SMC diseases
Geometrical and dimensional parameters are often used to define the state of human vasculature24,27 and link to vascular disease.47,48 Yet, the number of parameters required to define complex geometries prohibits rigorous characterization and produces a vast array of dimensions and possible interactions. We propose an alternative strategy that integrates dimensions and physiology into a single flow parameter based on the extent of recirculation in the vessel under study. Instead of limiting the evaluation of risk to a specific number of factors at specific locations in the vessel, we scanned the complete architecture of the vessel tree and determine the risk using a single variable that reflects how geometry affects recirculation. Our data suggest that the distinct geometry of each patient's vascular tree dictates different flow regimes, and that these regimes are the critical players in defining each patient's vascular phenotype. The coronary models, however, did not include the effect of forces caused by the myocardium during systole and diastole. Such forces further increase the oscillatory shear index in the LAD, but do not alter the localization of the recirculation sites.49
Indeed, Ox-LDL uptake by EC and monocyte adhesion to activated EC correlated directly with the extent of recirculation in each branch of the studied bifurcations. Furthermore, p-eNOS and KLF-2 expressions were higher in regions of lowest recirculation. The implication of these findings is that endothelial cells function differently when exposed to flows with different levels of recirculation.
The full sequence of atherogenesis was activated in regions of flow recirculation and stagnation. Recirculation was also a powerful predictor of SMC biology where TF expression tracked with the amount of recirculation precisely. In this context, the two elements of the paper come together nicely to explain the power of flow: arterial flow is protective of vascular injury and inductive of repair but subject to disruption by recirculation. Atherosclerosis and thrombosis are, however, highly multifactorial events. The inter-patient variability due to disease progression or after any of a range of vascular interventions can also originate from procedure trauma and release of anti-proliferative drugs, both responsible of potential thrombosis. Micro-recirculations caused at strut level by stent thickness and malapposition also increase the risk of thrombosis.26 In any case, restoration of flow after a stent deployment should reduce the risk of thrombus linked to blood recirculation.
In vivo correlations between flow recirculation and markers of vascular damage have been established in the past. In 2004, Richter et al.21 proved that regions of recirculation in a porcine model of the ilio-femoral bifurcation correlated with leucocyte recruitment and intimal hyperplasia levels. Gimbrone50 reported analogous findings in murine and rabbit aortic arches in 2010. Very recently, Assemat et al.51 found comparable results correlating in vivo atheroma with regions of bi-directional low shear stress in an aortic murine branch calculated by CFD. In this work, we tested the presence of macrophage in carotid bifurcations in mice fed with a high-fat diet. Regions of flow stagnation near the vessel walls were able to predict regions where macrophage recruitment was more dynamic.
4.3 The arterial replication platform is a multidisciplinary tool to study the local impact of flow disruptions
Ever since Gimbrone et al. designed the first in vitro flow chamber, researchers have tried to find accurate models of arterial flow disruptions. Though precise control over flow and geometry makes these models excellent tools for basic research, these systems do not generally simultaneously expose cells in a single construct to different flow regimes. Single-channel ex vivo and in vitro models are limited to one flow regime per system and lack the spatial resolution of different regions of a bifurcated artery.
Our system now allows exact replication of vascular architecture,10 simultaneous exposure of vascular cells to a physiological range of flow regimes within the same construct and quantitative measure of cell- and flow-specific responses. We can examine local recirculation and compare response of genetically identical cells,2 exposed to the same solutes and <1 cm apart. Cells that might be thought to be identical can now be shown to express significantly different levels of disease biomarkers under the control only of different degrees of recirculation. This spatial control over stimulus is critical in examining the cellular response to local flow alterations. While all inflammation and thrombogenic biomarkers generally rose when cells were exposed to oscillatory flow and static conditions, these values showed a clear correlation with the local extent of recirculation for each branch on each arterial bed. The three-dimensional approach localizes better the impact of flow patterns, but poses novel challenges at the same time. The studied biomarkers presented levels near the detection thresholds of the analytical methods applied, especially in small sections like recirculation regions of the bifurcated constructs.
Integrated frameworks such as the one we described here could be useful in basic research and clinical practice. As an in vitro tool, the platform allows for parametrical and mechanistic studies of the impact of patient-specific features in biological outcomes, not only for inflammation or coagulation but also for angiogenesis or cellular migration among many. Our platform has already been proved useful to determine the thrombogenicity and impact of different anti-proliferative treatments on endothelial recovery. The findings might well suggest a different focus in performing procedures—one that focuses on restoration of intact fluid flow rather than where stent struts are located. Increasingly flow is emerging as a quantifiable interventional parameter, and our results will place flow within a greater context. One could even envision extending this work to other flow domains such as the respiratory or urinary tracks.20 In the future, a parameter such as NVV may be provided to directly guide intervention based on individual patient state and vascular geometry of specific patients to prevent flow- and drug-related post-implantation complications.
4.4 Conclusion
Vascular cells attain a range of phenotypes that are most responsive to their flow environment. Endothelial health is preserved by arterial flow, but jeopardized in regions of flow recirculation in a quasi-linear manner. Similarly, smooth muscle cells exposed to flow are more thrombogenic in large recirculating regions. Thrombotic and inflammatory biomarkers correlate with the extent of recirculation that vascular cells are exposed to when lining certain vascular geometries. The integration of engineering of fluidics with vascular biology is the only means to examine these complex interactions.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
This work was supported by the National Institute of Health/National Institute of General Medical Science (RO1/GM049039 to E.R.E.); Ministerio de Ciencia e Innovación (BFU 2009-09804 to M.B., J.J.M., and E.R.E.); MIT International Science and Technology Initiatives (MIT-Spain Seed Fund 2010 to E.R.E. and J.J.M.); Generalitat de Catalunya (2013FI_B2 00093 to J.M. and MOBINT 2011 to P.S.), Banco Santander (Fórmula Santander 2011 to P.S.) and POSIMAT (PS2010-2013 to M.B.).
The authors thank the following people and institutions for their support: Philip Seifert for scanning electronic microscopy and histopathology at CBSET, Nicki Watson and Wendy Salmon for spinning confocal disk microscopy at Whitehead Institute, and Dr Jeffrey Wyckoff for multiphoton imaging at Koch Institute. We also acknowledge Dr Caroline C. O'Brien, Dr Vipul Chitalia, Dr Andrés A. García-Granada, Jay Wang, Elisabet Rosàs, and Fernando García-Polite for their technical support and intellectual discussion.
Conflict of interest: none declared.
- Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: firstname.lastname@example.com


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Fan of the show? Then it's imperative that you check out NCIS' latest mobile game and use these developer created tips to get you through it.
Pokemon Go Plus allows players to catch Pokemon with the press of a button, but can you use it to catch Incense spawns? How does that work?
In Pokemon Go, it is now finally possible for players to pinpoint where they caught a Pokemon thanks to a new update. How can you access this feature?
Pokemon Go has been receiving frequent app store updates, but when will the next one be out? And how often will these updates be released?
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The new Pokemon Go update allows players to see where a Pokemon was caught, but many are already complaining that the feature does not work properly.
Does the new Pokemon Go capture location feature show where eggs were found or where they hatched? Find out all the details here.
How does Pokemon Go's new capture location feature work? Is it anything like the maps we used to have? See photos and details about catch location here.
The new Pokemon Go update shows capture locations and lets Plus players catch incense spawns. But when will the new version be available for iOS and Android?
Niantic has released a new Pokemon Go Update: Version 1.9.0 for iOS and 0.39.0 for Android. Find out what's new in this latest version. What's different now?
Can you get extra bonus candy when you're walking your Pokemon Go Buddy? We know how many kilometers you need to walk, but how many candy will you get? Find out here.
In Pokemon Go, Tauros, Kangaskhan, Mr. Mime and Farfetch'd are exclusive to specific parts of the world. But can they be hatched from eggs anywhere?
How many kilometers do you need to walk a Pokemon Go Buddy in order to get enough candy to evolve it? See our list with the details here.
Become a master of shinobi arts by using this developer created tips guide for NARUTO SHIPPUDEN: Ultimate Ninja Blazing.
In Pokemon Go, it's important to know how much damage a move does before taking your Pokemon into a gym. This chart shows the DPS for every move in the game.


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Home \ Statements and speeches \
Statement by the Russian Ministry of Foreign Affairs regarding the international investigation into the causes of the Malaysian plane crash in the Ukrainian air space
As is common knowledge, on the 21 July the UN Security Council unanimously adopted a resolution regarding the holding of an international investigation into the crash of the Malaysian Boeing in Ukraine. The resolution, in particular, contained an appeal to all the fighting parties to stop military action in the area of the plane crash immediately and to provide access to this area to investigating bodies, the OSCE special monitoring mission in Ukraine and other international organisations, which are providing aid in the investigation.
In violation of this request by the Security Council, Kiev announced in public that it intended to take control of the plane crash area using arms and started a military attack in the area. Representatives of the Donetsk-based international investigation group attempted to reach the crash area and start their work several times.
On the 28 July, the Russian delegation drew the attention of its UNSC partners to this evident fact of violation of the Council's requests by Kiev and proposed to agree a press release in the Security Council reminding everyone of resolution 2166, primarily paragraph 7, where an immediate stop to military action in the area of the plane wreckage is requested. Unfortunately, our initiative was shamelessly blocked by US, UK and Lithuanian representatives.
This is another attempt to white wash over the Kiev authorities' actions, which prevents a truly independent and impartial international investigation into the circumstances of this tragedy.
29 July 2014


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text: Drake's 'Nothing Was The Same' By The Numbers By XXL Staff September 16, 2013 4:04 PM SHARE TWEET EMAIL REDDIT EMAIL REDDIT Number Of Times He Says "Me": 83 Previous Page Next Page Filed Under: By The Numbers | Drake | Lists | nothing was the same Category: News SHARE TWEET EMAIL REDDIT More Leave a comment EMAIL Print this page Pin it! REDDIT Share on Tumblr! Recommended For You ZergNet Around the Web ZergNet Best of XXL 5 Rappers Who Were Born in Obscure Cities Here Are All the Hip-Hop Albums That Topped the Billboard 200 in 2016 Top 5 Sneakers Coming Out This Weekend Including Adidas Yeezy Boost 350 V2, Air Jordan 5 Retro Bronze and More 16 Rappers Who Are Libras


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Latin, chines, and Arabic are different languages for the communication between the people in different countries.
Hence, the options (b), (c), and (d) are wrong.
The language of science is mathematics. Though the science can be expressed in Arabic and Latin, using mathematics it is possible to understand the science. The equations of science provide expressions of relationships between concepts.
Hence, the correct option is .


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Hotels near St. John's Cathedral
Search hotels near St. John's Cathedral in Kuala Lumpur
Trending now in Kuala Lumpur
- Most bookedArenaa Mountbatten Boutique Hotel
36 people looked at this hotel in the last hour
Most recent review
"A good hotel for families for the price bracket as you get two separate rooms. Some of the fixtures need fixing and the rooms can occasionally smell of cigarettes which seems to come from the ac. Great location for food with fab Indian opposite."
A Traveller, Aug 2016, GB
34 people looked at this hotel in the last hour
Most recent review
"The location was great. We can reach to MC Donald, KFC, Burger King in 5 minutes by walk. Also you can find local restaurants near by. But, when night you will see some people sleep on the street way to hotel. But, overall about the hotel was great."
i, Sep 2016, US
32 people looked at this hotel in the last hour
Most recent review
"I have a lovely stay. But I need to take some step up o the reception. Poor architectural planning."
A Traveller, Nov 2015, SG
28 people looked at this hotel in the last hour
Most recent review
"clean, friendly and helpful staff, fast check in and out, nice shower, good rooftop with a nice view . good Aircon, rooms are exactly as you see in the photos."
A Traveller, Sep 2016, GB
27 people looked at this hotel in the last hour
Most recent review
"I got a very clean room with a window. The hotel staffs were very nice to me. One staff carried my luggage to the bus station. The cleanliness of this hotel that brings me back there, always !"
A Traveller, Jul 2016, MY
20 people looked at this hotel in the last hour
Most recent review
"My stay was good, however if you are traveling alone... be aware to go out by yourself... had a bad experience on my return from twin towers but nothing happen... wifi inside of the rooms poor..."
A Traveller, Sep 2016, SG
- ML Inn HotelNo Guest Reviews yet
14 people looked at this hotel in the last hour
9 people looked at this hotel in the last hour
- Reggae Mansion SuiteNo Guest Reviews yet
3 people looked at this hotel in the last hour
- NIDA Rooms Masjid Jamek Tun PerakNo Guest Reviews yet
Map of hotels near St. John's Cathedral
- Petronas Twin Towers
- Kuala Lumpur City Centre
- Berjaya Times Square
- Suria KLCC Shopping Centre
- Aquaria KLCC
- Sunway Lagoon Theme Park
- Pavilion Kuala Lumpur
- University of Malaya
- Mid Valley Mega Mall
- Sunway Pyramid Mall
- KLCC Park
- Sepang International Circuit
- 1 Utama
- Petaling Street
- First World Plaza
- Putra University-Malaysia
- Merdeka Stadium
- Kuala Lumpur Convention Centre
- Batu Caves
- Genting Highlands
Hotels in Kuala Lumpur near St. John's Cathedral
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Todays info was in Alterra's 10-Q in August - it was public info then and really had no effect on the stock price (these residences always were step children for both companies and now they can convert back to being HCR residences - from what I remember some of them resembled remodeled Holliday Inns (do you HCR history research) - in any matter they were not typical Alterra residences.How does it effect things? The Debt and assets were reduced.The HCR law stuff? - Is also old news - so the price if it changed any already reflects every bad news item out there. There is only one last piece of bad news - that can effect the price - will Alterra get its head above water? - the next 1/4 ly should give us a pretty good idea or hint.The only reasons to hold the stock - Too high a basis to sell (sales commission takes away basis)Company gets bought out (good? don't know)Company gets some kind of restructured debt.They turn things around a make enough to service the debtThe interest rates effect debt service ability.And THEN There are the reasons not to - You could lose every last cent you have in the stock.


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Skip to comments.Alan Dershowitz: I Would Find Zimmerman 'Not Guilty'
Posted on 07/10/2013 5:43:17 AM PDT by SJackson
The verdict is in from Alan Dershowitz: if the renowned Harvard Law professor were on the jury hearing the George Zimmerman murder trial, he would find the defendant not guilty.
"I would say there's reasonable doubt. I would say nobody knows who started the initial fight," Dershowitz told "The Steve Malzberg Show" on Newsmax TV.
"Remember, it's monumentally irrelevant who's morally guilty here."
Zimmerman, who identifies himself as Hispanic, is charged with second-degree murder in the death of Trayvon Martin, an unarmed teenager in Sanford, Fla.
Zimmerman, a 29-year-old neighborhood watch volunteer for his community, says he shot Martin, 17, in self-defense during an altercation. But prosecutors charge that Zimmerman profiled the African-American youth as he walked back from a convenience store to the home of his father's fiancée.
"Whether or not Zimmerman was a racist and racially profiled and shouldnt have been doing it and didnt listen to police, that's all irrelevant in Florida law," Dershowitz said.
"The case begins when the first blow was struck, essentially. And we dont know who struck the first blow. We dont know if Trayvon Martin came out from behind of a dark area and jumped him and got him down.
(Excerpt) Read more at newsmax.com ...
Writer is in the correct.
Alan is obviously a racist bigot.
If I were a juror I would find him not guilty too and my verdict would have nothing to do with the law. (it doesn't need to)
My not guilty verdict would be a direct result of attempts to intimidate and threaten a guilty verdict out of the jury and it would be a direct result of the media driving the attitudes.
If more jurors knew they could do this we might finally put a stop to the media circus that sets up camp around trials they decide are high profile.
The question I have is, did Trayvon Martin have a "legit" reason to even be in the area, because if my memory is correct, that was a gate community?
In other words, was he in the process of getting ready to do "b and e" or "breaking and entering" ?
When an armed man and an unarmed man get involved in fisticuffs, you can pretty much assume the unarmed man started it. There are a lot of reasons for this.
It doesn't matter if he profiled someone or threw the first punch or whatever. Had the prosecution somehow destroyed the theory that Martin was atop Zimmerman then it can get confusing. But they didn't.
This shouldn't even go to the jury; it should rightfully be ruled from the bench and the Florida state portion of this circus should be over with. Alas, it will likely be followed by some fast and furious announcement from Eric Holder of federal civil rights case being filed against Zimmerman.
I'd say Zimmerman's injuries go a long way toward answering that question.
The state was literally forced into indicting Zimmerman by the media in the first place. I wonder if, given the miserable job they're doing, the prosecution was coached to throw the case rather than take the chance of a conviction?
Me too. He had to be arrested and charged so the Martins and the politicians could reap the benefits. Hope he gets off and sues every you know what and becomes the richest guy in the world.
Does it matter who started the fight? I disagree. I say it does matter. The argument that the answer is no is that even if Zimmerman had started a fight (meaning, a fist-fight), he had no avenue of escape at the time he pulled the trigger and, being in fear of his life, was justified in using deadly force. This would justify a lot of homicides by people who start fights and find, at some point, they're getting beaten. I would say, however, that this construction of the crime wouldn't be murder in the second degree, it would be manslaughter.
The real point is that we don't know who started the fight. The burden is on the prosecution to prove their case beyond a reasonable doubt. As to what the prosecution is trying to prove, I actually have no idea. During the trial, the prosecution has said they have multiple theories of the crime., meaning they themselves are not convinced by any one theory of the crime beyond a reasonable doubt. According to one of their theories of the crime, Trayvon was on top and was attempting to pull away from Zimmerman at the time he was shot. Yet, they do not have a shred of evidence proving that Trayvon was attempting to pull away. Therefore, it is reasonable to believe, since the prosecution believes, that Trayvon was on top. And, in the absence of any evidence that Trayvon was pulling away, it is reasonable to believe he wasn't pulling away. Indeed, via Di Maio's testimony, it is unlikely that Trayvon was pulling away as the hole in the clothing would not have lined up with the hole in the young man's body, if he had been trying to pull away and was being restrained by Zimmerman holding onto his clothing.
So, the verdict should be not guilty.
As for the controversy about whether Trayvon's interest in guns and in fighting should be allowed in court, if the prosecution would stipulate as to what theory of the crime they are arguing, the evidence might not matter. As it is, the evidence of fight should come in, since the prosecution contends that Zimmerman was not being beaten in any serious way, and the jury needs to be able to compare the relative fighting abilities of the two. On the other hand, under no theory of the crime is the gun evidence relevant. And, in my own opinion, interest in guns and in fighting by young men are good things. Back in the day, I was proud to have young men with a fighting spirit in my unit and felt entirely confident that I and my NCOs could discipline them.
Never thought of it that way, but you're right cuban... Insightful.
I have the feeling that even if there was video evidence of Martin striking the first blow, and video evidence of him on top of Zimmerman, pounding away, the political pressure on the state to bring charges would've still been strong.
Too bad one of Obama's drones wasn't overhead at the time and captured the events.
For all we know, there was.
He was staying at his father's girlfriend's apartment in the complex.
But, according to none other than Rachel Jeantel, poor little Trayvon made it safely all the way to that apartment, then went back out to confront Zimmerman.
Reading between the lines of her testimony, Rachel Jeantel is the reason Trayvon Martin is dead.
She was on the phone talking trash to him about the "creepy-#%% cracker rapist" following him and how if he was a real gangster he'd give the cracker a beatdown he'd never forget.
When Trayvon was killed because of her she went dark.
It wasn't until Benjamin Crump hunted her down and gave her a script to follow that she resurfaced.
Case closed.
I hope the Defense uses your exact words.
Thank-you for your answer and now I have it all figured out. It was a planned attack on George Zimmerman.
She could very well get in trouble herself. Surprised she is not in trouble.
"Reading between the lines of her testimony, Rachel Jeantel is the reason Trayvon Martin is dead."
The reason Trayvon died is because he had two lousy parents. Had the parents provided proper supervision and guidance he would not have been suspended from school and therefore would not have been in Sanford that night. Alternatively, if Trayvon's father had been at home that night with the boy, instead of running around with his "fiancé", Trayvon wouldn't have been wandering around in the rain talking to Jeantel. Any decent parent would have grounded a teen suspended from school and taken his cell phone away. If he had been my kid, he'd have either been sitting at his desk in his room studying or doing chores around the house under my direct supervision.
I was just reading between the lines, but the fact is that Trayvon did make it home safely and went back out.
What a bizarre, distancing turn of phrase.
It's as if he were writing "Sharpton, who identifies himself as Finnish" in order to show how unbelievable the subject's statement is to the author.
Good logical post.
He'll be found not guilty. The verdict isn't the problem. It's the rioting that will be the problem.
When an armed man and an unarmed man get involved in fisticuffs, you can pretty much assume the unarmed man started it. There are a lot of reasons for this.
I might modify that to "when an armed man, and especially one who is legally armed..."
Legally armed people tend to be very law-abiding. They don't generally go around starting fistfights.
Armed men who are about to get into a fistfight also don't call the police first.
Armed men don't generally start a fistfight, because they know it could end with them having to use their gun on the other person. I dare say that even if you don't care that much about the other person's life, generally speaking, no legally armed person wants to be known as "the guy who killed so-and-so."
Nor does any legally armed person want the hassle and danger and expense of being arrested and tried for murder.
Finally, if someone with a gun really wants to harm another person, he's not going to use his fists to do so.
You're right on every count Jeff...
With what little the prosecution had in terms of evidence, the prosecutors didn't need to throw the case. Their most zealous efforts would hardly be distinguishable from throwing it.
Dershowitz is correct and understanding this concept is monumentally important to understanding this case. Those in the Martin camp have sought to characterize Zimmerman as morally guilty and therefore he is entitled to use less force to defend himself. In other words, if, they imply, Zimmerman contributed to an atmosphere which resulted in a fight which entitled Zimmerman resort to self-defense he was morally guilty and therefore not entitled to use deadly force. That is to say, the degree of force used was unreasonable in the sense that it was immoral.
When Dershowitz makes his observation that it is irrelevant who is morally guilty he is making the distinction which I have been trying to make in many posts, that the right to employ self defense is independent of these moral considerations. The right to use self-defense depends on the defendant's "imminent apprehension of serious bodily harm" and that apprehension must be "reasonable." But that test of reasonableness has nothing to do with the defendant's morality, it has only to do with whether his assessment of imminent serious bodily harm is accurate in the sense that it is credible. The test of reasonableness is designed to elevate a test from mere subjectivity on the part of the defendant to a standard which jury can assess according to the circumstances in the case having to do with the imminence and seriousness of the harm.
Significantly, once the jury concludes that the defendant had the right to resort to self defense, he automatically has the right to resort to deadly force, regardless of his morality.
I have said this in the context of the jury arriving in a compromise verdict finding Zimmerman guilty of manslaughter because they are led down the false path of morality. The simple fact is that under the law in Florida if Zimmerman is innocent of murder II because he properly resorted to self-defense, then he is automatically innocent of the charge of manslaughter for the same reason.
I have said the judge should charge to this effect to avoid any improper compromise verdict which the jury has no right to make.
I still am having a really tough time with the term 'unarmed' applied to Trayvon Martin.
IIRC, the can of Arizona Iced Tea which he had bought along with the Skittles is a 20 OZ can.
I just browsed my cupboard of canned goods.
I have cans of DOLE crushed pineapple—they are all 20 OZ.
I picked up that can- hefted it a few times & swung it in a motion which would become an overhead blow to someone or something.
Then I asked myself:
IF someone was using that 20 OZ can to hit me about my head & upper body, would I consider that a WEAPON?
Hell, yes!!!!!
Zimmerman, who identifies himself as Hispanic, is charged with second-degree murder in the death of Trayvon Martin, who identifies himself as worm food.
If Z is found guilty, I bet America will rise up against the injustice. People will tolerate a guilty person who is found not guilty; but we will not tolerate the injustice of an innocent man found guilty. At least I hope that's true. Americans need to stand up against our justice system
Doesn't answer who threw the first blow, but certainly who threw the effective blows, and who might have been in fear of his life.
I haven't had time to spend on these threads as I'd liked, so maybe this has been answered. But it's my understanding that if Zimmerman initiated, on the one hand he could well have felt fear for bodily harm and legitimately defended himself, however the initiating actions could open him to lesser charges based on negligence and/or imprudent action. Of course the prosecution, in their wisdom, didn't address those possibilities. I'm not suggesting Zimmerman is culpable for those actions.
Do we agree than contributing actions could lead to criminal liability? If Zimmerman started the fight, and came out on the short end, legitimately defending himself isn't murder, but there might be something there. Not suggesting that happened.
If I sell a firearm to someone without documentation, required or not, I could be subject to civil or criminal penalties.
If I sell a firearm to known criminals, who use it to kill anyone from a Border Patrol agent to a Police Chief to hundreds of citizens, many probably criminals, of another nation, I'm guilty of nothing.
Go figure.
Yes, I don't remember Zimmerman identifying himself as anything. Some corners of the media identified him as Jewish. I wonder what he identifies himself as, or why it matters.
A different more serious problem should it come to pass.
You present the moral/legal distinction quite well, though in this situation I don't see the conflict I suspect Alan might. As to lower charges, though there could be something there in the context of imprudent or negligent action which I don't see, they weren't charged, thus depriving Zimmerman of the ability to defend against them. They shouldn't be an issue.
A few people with an Arizona Tea to put in their sock would have been well armed on 9/11. Able to defeat box cutters. Couple bars of soap works too. Camera on a strap.
The first blow is the one most likely to land - Martin didn't have a mark on him. That makes the probability overwhelming Martin threw the first blow. End of story.
Clearly the probability.
On the matter of civil liability, the standard is preponderance of evidence (or, who has the most). As we know from the OJ trial, you can meet this standard and not meet the standard for criminal conviction of beyond reasonable doubt. But, what money could they get from George Zimmerman? Even if he were found liable, he'd simply declare bankruptcy and walk away. He wouldn't even need a lawyer. Just defend yourself pro se and let the other side spend hundreds of thousands of dollars for the possibility of nothing.
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