Review
Advances in endothelial shear stress proteomics Expert Review of Proteomics Downloaded from informahealthcare.com by Nanyang Technological University on 04/24/15 For personal use only.
Expert Rev. Proteomics 11(5), 611–619 (2014)
Sabika Firasat1,2, Markus Hecker3, Lutz Binder1 and Abdul R Asif*1 1 Institute of Clinical Chemistry UMG-Laboratories, Robert-Koch Str. 40, 37075 Goettingen, Germany 2 Department of Biosciences, University of Wah, Quaid Avenue, Wah Cantt, Pakistan 3 Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany *Author for correspondence: Tel.: +49 551 392 2945 Fax: +49 551 391 2505 [email protected]
The vascular endothelium lining the luminal surface of all blood vessels is constantly exposed to shear stress exerted by the flowing blood. Blood flow with high laminar shear stress confers protection by activation of antiatherogenic, antithrombotic and anti-inflammatory proteins, whereas low or oscillatory shear stress may promote endothelial dysfunction, thereby contributing to cardiovascular disease. Despite the usefulness of proteomic techniques in medical research, however, there are relatively few reports on proteome analysis of cultured vascular endothelial cells employing conditions that mimic in vivo shear stress attributes. This review focuses on the proteome studies that have utilized cultured endothelial cells to identify molecular mediators of shear stress and the roles they play in the regulation of endothelial function, and their ensuing effect on vascular function in general. It provides an overview on current strategies in shear stress-related proteomics and the key proteins mediating its effects which have been characterized so far. KEYWORDS: endothelial dysfunction • mass spectrometry • proteomics • shear stress
Vascular endothelium & shear stress
The endothelium lines the whole circulatory system from the heart to the smallest capillaries. However, endothelial cells (ECs) from different vascular sites are heterogeneous both in morphology and with respect to their protein expression and surface markers. This heterogeneity contributes to their physiological diversity across the vascular tree [1]. ECs regulate vascular homeostasis, tone and permeability; affect the balance between coagulation and fibrinolysis; contribute to inflammatory reactions; affect cellular propagation in the vessel wall; and interact with other cell types, for example, vascular smooth muscle cells, platelets, leukocytes, retinal pericytes, renal mesangial cells and macrophages through the production of different chemical mediators [2]. The inability of ECs to adequately carry out any of these functions is referred to as endothelial dysfunction, which is a hallmark of most forms of cardiovascular disease [3–5]. The endothelium is constantly exposed to hemodynamic forces due to pulsatile blood flow and pressure through the vasculature [6]. These hemodynamic forces include hydrostatic pressure created by the hydrostatic forces of blood within the blood vessels, cyclic stretch produced by longitudinal forces acting on the cell during vasomotion and shear stress, which informahealthcare.com
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are transduced between the ECs by defined intercellular connections, and the dragging frictional force applied tangential to the apical membrane of ECs by the blood flow [7]. Among these, cyclic stretch and shear stress are fundamental to modulating endothelial physiology, yet both make rather distinct and differing contributions to vascular disease [8–11]. Shear stress is of primary importance since it elicits a biochemical signal response in ECs [7,12]. The EC surface harbors numerous potential mechanoreceptors, for example, integrins, ion channels, receptor tyrosine kinases (such as vascular endothelial growth factor receptor, heterotrimeric G proteins and platelet-EC adhesion molecule-1 as well as supramolecular structures (or some of their constituents) such as the primary cilia, the apical glycocalyx, cell–cell junctions and the cytoskeleton, which, together with fluidity changes in the membrane lipid bilayer, aid in sensing or transmitting the initial biomechanical stimulus. These surface receptors or structures act as transducers of the flow-induced shear stress in ECs and trigger a complex network of interconnected intracellular pathways leading to adaptive changes in gene expression, cell metabolism and cell morphology [13–22]. Fluid shear stress is not uniform across the vascular system. The magnitude of shear stress ranges from 1 to 6 dynes/cm2 in the venous
2014 Informa UK Ltd
ISSN 1478-9450
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Table 1. Summary of proteomics studies on endothelial cells subjected to shear stress in vitro.
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Shear stress
Ref.
Type
Magnitude (dynes/cm2)
Exposure time
Proteomics strategy
Apparatus
In vitro model
Laminar
12–15
1 min, 2.5 min, 10 min
IMAC, SPE-CE-MS/MS
Cone and plate viscometer
BAECs
[58]
Oscillatory
0 ± 3.0
4h
LC–MS/MS
Dynamic flow system
BAECs
[93]
Laminar
15
10 min, 3 h, 6 h
ICAT, LC–MS/MS
Flow loop perfusion apparatus
BAECs
[50]
Laminar
10
18 h
ICAT, LC–MS/MS
Cone and plate viscometer
RVECs
[70]
Pulsatile oscillatory
23 0.02 ± 3
–
LC–MS/MS
Dynamic flow system
BAECs, HAECs
[82]
Laminar
25
30 min
DIGE, LC–MS/MS
Parallel plate flow chamber
HUVECs
[63]
Laminar
15
10 min, 3 h, 6 h
iTRAQ, LC–MS/MS
Parallel plate flow chamber
BAECs
[68]
Laminar
30
24 h
2DE, LC–MS/MS
Cone and plate viscometer
HUVECs
[54]
Laminar
5 12
6 h, various
LC–MS/MS
Parallel plate flow chamber
BAECs, HEK293, HUVECs
[57]
Laminar Oscillatory
15 8
24 h
SILAC, LC–MS/MS
Cone and plate viscometer
HUVECs
Steady
18 h
ICAT, LC–MS/MS
Cone and plate viscometer
RVECs
[83]
Pulsatile
10 and 30 5–15
Pulsatile
12 ± 4
1 h, 2 h, 4 h, 8 h
LC–MS/MS (PTM)
Dynamic flow system
HUVECs
[84]
[100]
BAECs: Bovine aortic endothelial cells; DIGE: Difference gel electrophoresis; HAECs: Human aortic endothelial cells; HEK293: Human embryonic kidney 293 cells; HUVECs: Human umbilical vein endothelial cells; ICAT: Isotope-coded affinity tags; IMAC: Immobilized metal affinity chromatography; iTRAQ: Isobaric tags for relative and absolute quantitation; PTM: Posttranslational modification; RVECs: Rat vascular endothelial cells; SILAC: Stable isotope labeling by amino acids in cell culture; SPE-CE: Solid phase extraction capillary electrophoresis.
network and from 10 to 70 dynes/cm2 in the arterial segment of the vasculature [23–26]. Straight parts of the vasculature experience steady and high laminar shear stress (LSS), which protects from atherosclerotic lesions [6,27,28]. On the other hand, at branch points and curvatures of the arterial system, blood flow is disturbed and wall shear stress is low, nonuniform and irregularly distributed [26,29,30]. This low and oscillatory shear stress (OSS) can cause EC dysfunction by inadequately modulating normal physiological EC signaling and gene expression, possibly leading to atherosclerosis and/or thrombosis [6,31–33], if accompanied by other risk factors that act on the vasculature, such as genetic predisposition, biochemical factors or lifestyle [34–36]. Proteomics of endothelial cells exposed to laminar shear stress
Proteomes are intricate and dynamic entities that are still poorly explored. Recent advances within the field of proteomics, including both global and targeted protocols, have 612
stimulated a transition from conventional protein identification to functional investigations, which will provide invaluable insight into disease pathogenesis [37]. The application of proteomic tools in the clinical setting is continuously increasing. However, relatively few studies have been investigating the proteome of ECs cultured under shear stress. Traditional techniques mainly based on immunological detection methods have identified some of the shear stress-responsive proteins [38–49]. But these techniques can identify only one protein at a time and heavily depend on the availability and quality of specific antibodies, limiting their feasibility for comprehensive analyses [50]. Using mass spectrometry-based strategies, however, the up- or downregulation of a large numbers of proteins in response to shear stress can be examined to disclose the molecular mechanisms contributing to endothelial dysfunction. Proteomic studies on cultured ECs involving the analysis of human umbilical vein ECs and ECs from various model species under different forms of shear stress are summarized in TABLE 1. Expert Rev. Proteomics 11(5), (2014)
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Endothelial shear stress proteomics
Endothelial dysfunction as an early sign of atherosclerosis is attributed to a decrease in nitric oxide (NO) bioactivity and enhancement in oxygen free radical formation. NO plays a major role in maintaining cardiovascular homeostasis as it is a potent vasodilator, antioxidant and anti-inflammatory mediator. It is synthesized by endothelial nitric oxide synthase (eNOS), and the most important physiological stimulus for eNOS expression is shear stress. Failure to adequately respond to this trigger for upregulating the production of eNOS should thus severely affect endothelial function. In fact a point mutation in the 5´-flanking region of the eNOS gene, -786T!C, has been reported to render the gene insensitive to LSS suppressing its transcription. As a consequence, human ECs homozygous for the eNOS CC gene variant are irresponsive to shear stress leading to endothelial dysfunction [51–54]. Severely reduced NO production, however, could indirectly affect the expression of other proteins in CC genotype cells. Asif et al. therefore compared the protein profiles of human umbilical vein ECs carrying the wild type (TT) or the mutant (CC) single nucleotide polymorphism after exposure to shear stress, using 2D electrophoresis and LC–MS/MS strategy. A total of 14 proteins were detected to be differentially expressed, of which eight proteins were similarly regulated in response to shear stress in both cell types. Four proteins involved in NO-dependent endoplasmic reticulum stress response, however, were upregulated by LSS in cells carrying the TT genotype. In contrast, cells carrying the CC genotype showed a unique increase in the transcription factor Egr-1-mediated expression of manganese-containing superoxide dismutase (SOD-2) in response to LSS. SOD-2 plays a critical role in protecting cells from superoxide, a key mediator of oxidative stress within the mitochondria. This increased SOD-2 expression in the CC genotype ECs may account for the fact that individuals homozygous for the -786T!C single nucleotide polymorphism do not show an earlier onset of coronary artery disease as compared to those carrying the TT genotype, as it may result in an antiatherosclerotic phenotype of these cells despite reduced eNOS activity, which in the long run, though, poses a higher risk after all [54,55]. eNOS activity is regulated by phosphorylation/ dephosphorylation of serine (Ser) and threonine residues by protein kinases/phosphatases [56,57]. Shear stress not only affects eNOS expression but also indirectly enhances eNOS activity via phosphorylation. To investigate the role of AMP-activated protein kinase in eNOS activation and NO bioavailability in ECs subjected to LSS, Chen et al. employed a nano-LC–MS/ MS-based proteomics approach. The study reported an enhanced AMP-activated protein kinase-dependent phosphorylation of eNOS Ser-633 in response to LSS, which acts as a functional signaling event for NO production, further underlining the role of LSS as a key activator of eNOS [57]. Gallis et al. also reported increased phosphorylation of eNOS in response to increased LSS in cultured bovine aortic endothelial cells (BAECs). Using immobilized metal affinity chromatography followed by solid phase extraction capillary electrophoresis and MS/MS analysis, two specific LSS-dependent eNOS informahealthcare.com
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phosphorylation sites, Ser-116 and Ser-1179, were identified. Activation of phosphatidylinositol 3-kinase and protein kinase B was found to be involved in shear stress-dependent eNOS phosphorylation and stimulation [58]. The stimulation of eNOS results in an increased production of NO, which, in turn, can interact with susceptible cysteine residues, resulting in S-nitrosylation of proteins. S-nitrosylation is an important posttranslational modification that plays a role in the modulation of cardiovascular function via the regulation of mitochondrial metabolism, intracellular Ca2+ handling, protein trafficking and cellular defense against apoptosis and oxidative stress [59–61]. Huang et al. analyzed the changes in S-nitrosylation of reactive cysteine residues present in endothelial proteins after the application of 25 dynes/cm2 LSS by the CyDye switch method (a modified biotin-switch method involving selective reduction of S-nitrosothiol using copper ions and ascorbate followed by in-gel detection of modified proteins by fluorescent cyanine dyes [62]). Using 2D fluorescence difference gel electrophoresis followed by MS/MS analysis, 12 proteins with a significant rise in S-nitrosylation after shear stress were identified. In addition, the S-nitrosylated cysteine residues of tropomyosin and vimentin were located in the hydrophobic motif of these proteins, leading to the suggestion that increased S-nitrosylation of these two cytoskeletal proteins is important for the adaptation and remodeling of ECs in response to laminar flow conditions [63]. Increased generation of endogenous reactive oxygen species is reported to cause cellular dysfunction in a number of pathophysiological conditions. The hyperglycemia-induced overproduction of superoxide appears to be the initial mechanism by which diabetes enhances reactive oxygen species generation in ECs. Hyperglycemia is thus involved in the abnormal endothelial response to shear stress via oxidative stress resulting in the impairment of cellular functions, for example, through nonenzymatic protein glycation, protein kinase C activation and modification of transcription factors [64–68]. The LC–MS/MS analysis of protein expression of BAECs pretreated with high glucose and subjected to LSS versus controls cultured at normal glucose levels revealed a set of differentially expressed proteins involved in structural integrity, biosynthesis and metabolism, cell proliferation and apoptosis, signal transduction and protein degradation, indicating a role for hyperglycemia in the development of a shear stress-mediated atheroprone phenotype of ECs [68]. These findings strongly indicate that shear stress is implicated in diabetes-related cardiovascular complications. To study the effect of different times of exposure to shear stress, Wang et al. established protein profiles of cultured BAECs by isotope-coded affinity tags labeling coupled with LC–MS/MS analysis. Out of a total of about 2000 proteins, 142, 213 and 186 proteins were regulated in response to 10 min, 3 h and 6 h of LSS, respectively. Among these were constituents of important signaling pathways such as integrins, G-protein-coupled receptors and glutamate receptors as well as elements of Notch, cAMP-mediated and PI3K/AKT pathways [50]. The PI3K/AKT pathway is involved in the activation 613
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of eNOS to produce high levels of NO in response to different stimuli, for example, growth factors, hormones [69] and shear stress. Using a labeled quantitative proteomics approach, Freed et al. analyzed the role of phosphatidylserine in the LSS-mediated protection of ECs against apoptosis [70]. Phosphatidylserine is known to activate protein kinases, which in turn control major signal transduction pathways of cell proliferation and cell survival [71,72]. They found that phosphatidylserine, which is present in the plasma membrane in ECs, is essential for the shear stress-mediated activation of the PI3K/ AKT survival pathway. These results further emphasize the role of shear stress in protecting ECs from apoptosis, which would be detrimental in atherosclerosis as it can lead to plaque rupture and thrombosis [73]. Fluid shear stress also contributes to the differentiation of ECs into a blood–brain barrier (BBB) phenotype that controls transport of biological substances required for brain metabolic processes and neuronal functions. The BBB consists of cerebral microvascular ECs characterized by the lack of fenestrations, few pinocytotic vesicles and the presence of well-developed tight junctions, which constitute a diffusion barrier to selectively exclude most blood-borne substances from entering the brain. In a proteome study using 2DE followed by densitometry analysis of a humanized dynamic in vitro BBB model, a significant upregulation of tight junction proteins in response to shear stress was shown. Thus, these observations highlight the role of LSS in promoting junctional integrity of ECs [74]. Besides its importance in neurodegenerative diseases [75,76], loss of endothelial cell–cell junction integrity at other sites of the vasculature, namely bifurcations and curvatures of large conduit arteries, is also associated with the development of atherosclerosis [77,78]. ECs in linear sections of the vasculature experience high LSS which is pulsatile in nature, with fluctuations in magnitude depending on the cardiac cycle [7]. In vitro studies often use a flow pattern that generates a temporally and spatially uniform, steady and positive shear stress. Nevertheless, there are some qualitative and quantitative differences in EC responses under pulsatile shear stress as compared to steady shear stress conditions [7,79–81]. In some recent studies, experimental set-ups were therefore modified to create pulsatile shear stress, for example, by utilizing dynamic flow systems [82–84]. Freed and Greene used a modified cone and plate viscometer to expose cultured rat vascular ECs to pulsatile shear stress to study the mechanisms associated with its prolonged defense of ECs against proapoptotic stimuli. The proteomic analysis of isolated TNF-a-associated signaling proteins using isotopic labeling quantitative proteomics revealed an increased expression of caspase recruitment domain-containing adaptor protein CARD9 in cells after treatment with steady and pulsatile shear flow prior to the exposure to apoptotic stimuli as compared to nontreated cells. In ECs, exposure to shear stress for 18 h conferred protection from TNF-a-induced apoptosis through an NO-independent mechanism that relied on de novo protein 614
synthesis [83]. CARD9 is a known activator of the NF-kB (nuclear factor kappa-light chain enhancer of activated B cells) pathway, which protects cells from apoptosis. These data suggest that laminar and pulsatile physiological shear stress contribute to the protection of ECs via NO production as well as NO-independent mechanisms. Pulsatile shear stress exerts antioxidative and anti-inflammatory effects on vascular ECs, in part by the induction of sirtuin 1 (SIRT1) [85]. SIRT1, a class III histone deacetylase, causes deacetylation of targets, for example, forkhead box O, peroxisome PPAR-g, PPAR-g coactivator 1a, NF-kB and eNOS [86,87]. To unveil the underlying mechanism involved in the flow-mediated upregulation of SIRT1, Wen et al. [84] identified phosphorylation sites based on nano-LC–MS/MS. Their study revealed that pulsatile shear stress induces an upregulation of SIRT1 level and activity by enhancing Ca2+/ calmodulin-dependent protein kinase kinase b (CaMKK-b) phosphorylation of SIRT1 at Ser-27 and Ser-47. The role of CaMKK-b in SIRT1 activation was further confirmed by the drastically increased atherosclerosis in mice lacking CaMKK-b or endothelial SIRT1 [84]. Proteomics of endothelial cells exposed to oscillatory shear stress
Shear stress experienced by ECs is not uniform throughout the vascular system, since it is a function of blood flow patterns across the vascular system produced by the cardiac cycle. In straight parts of vessels, blood flow exerts a high, directed LSS on ECs with fluctuations in magnitude depending on the phase of the cardiac cycle, whereas at branches and curvatures, laminar blood flow is disrupted resulting in separated flow patterns that generate a low, nonuniform OSS. At such locations, it is probably the lack of LSS as compared to the relatively moderate rise in OSS that is responsible for the observed change in phenotype and gene expression [7]. In addition, due to the reflection of the pulse wave at the bifurcation, ECs located at the outer vessel walls become cyclically stretched. This may be a factor that is equally important as the sharp drop in LSS promotes this phenotypic shift. As a consequence of the endothelial increase in proinflammatory gene expression, branches and curvatures of large conduit arteries are particularly prone to develop atherosclerotic lesions (atherosclerosis predilection sites) [88–90], as was shown in normal pigs and transgenic mice [91,92]. Moreover, the disturbed flow adds to postsurgical neointimal hyperplasia, in-stent restenosis, vein bypass graft failure, transplant vasculopathy and aortic valve calcification. In the venous system, disturbed flow due to reflux, stasis and outflow obstruction causes venous inflammation and thrombosis leading to the development of chronic venous diseases [25]. It is well known that before menopause women seem to be better protected against the progression of atherosclerosis than men, and this most likely is related to the level of estrogen. A protein profile established by LC–MS/MS of BAECs Expert Rev. Proteomics 11(5), (2014)
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Endothelial shear stress proteomics
pretreated with 17b-estradiol (E2) followed by exposure to OSS [93] showed that treatment with the hormone resulted in the downregulation of OSS-mediated NADPH oxidase 4 expression and superoxide production as well as in the upregulation of eNOS expression and NO production. The extent of low-density lipoprotein (LDL) modifications was also decreased in the presence of physiological concentrations of E2 under static as well as OSS conditions. Moreover, the LC–MS/MS analyses of xanthine oxidase-mediated protein nitration of LDL revealed that pretreatment with E2 reversed nitration (posttranslational modification) at the tyrosine residue 2535 of the a-2 helix domain. These data suggest that the effect of E2 is mediated, in part, by the downregulation of NADPH oxidase 4 and the upregulation of eNOS, leading to a suppression of oxidant production, which may explain the proatherogenic impact of OSS on arterial bifurcations. Furthermore, this study may explain how the indirect antioxidant activity of E2 contributes to the aforementioned gender-specific cardioprotection [94,95]. Ai et al. [82] analyzed the pathophysiological significance of SOD-2 in response to pulsatile and OSS using LDL particles to assess protein nitration via peroxynitrite (ONOO-). The analysis of apolipoprotein B-100 (apoB-100), the protein component of LDL, by LC–MS/MS revealed that OSS increased whereas pulsatile shear stress significantly decreased the extent of LDL protein nitration in comparison with static controls. OSS is a potent stimulus for oxidative stress, whereas SOD-2 is an important dismutase of superoxide anions (O2-) derived from the respiratory chain acting on the mitochondrial matrix [82]. OSS favors ONOO- formation through alteration of the O2- to NO ratio, leading to protein-bound nitrotyrosine formation [96–98]. Nitrotyrosine is prominently present in atherosclerosis-prone, OSS-exposed regions of the vasculature where SOD-2 is diminished, indicating implications of spatial variations in SOD-2 expression by specific shear stress patterns in atherosclerosis and inflammation [82,96,99]. In the abovementioned proteome studies, the impacts of either LSS- or OSS-induced regulatory or damaging effect(s) on ECs are compared with the situation under static conditions. Burghoff and Schrader [100], however, analyzed the secretome of ECs under static and shear stress (both LSS and OSS) conditions using a quantitative proteomics approach by stable isotope labeling of amino acids in cell culture. A total of 240 secreted proteins were identified in all three secretomes combined (under static, LSS and OSS conditions). Of these, 101 were differentially regulated under shear stress, highlighting the impact of shear stress on the contribution of ECs to the regulation of vascular homeostasis [100]. A subset of eight proteins specific for LSS and one comprising five proteins specific for OSS were detected, emphasizing dissimilarities of laminar and oscillating flow conditions. Most in vitro experimental models mimicking in vivo flow conditions have been used to study cellular responses to LSS. Few studies applied modified flow systems to create low, oscillatory flow which is experienced informahealthcare.com
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by ECs present at atheroprone regions of the vasculature [82,93,100]. However, a disadvantage of these modified systems is that experiments cannot be performed in parallel unless multiple devices are available. Another drawback is that acute effects of shear stress on ECs cannot be investigated due to the technical difficulties of subjecting a large number of cells to defined fluid flows over prolonged periods of time under sterile conditions. Some recent studies have proposed the use of orbital shakers to correlate shear stress with cellular responses to study many cases simultaneously [101–103]. However, to our knowledge, no such analysis of the ECs proteome in response to orbital shear stress has been published so far. One reason could be that it is difficult to precisely calculate the (heterogeneous) shear stress exerted by the rotating fluid on the EC monolayer – an obstacle that is likely to be overcome by applying computational fluid dynamic solvers in the future [102,103]. Expert commentary
Proteomic studies represent the current frontier strategy to understand disease mechanisms and identify novel diseaseassociated markers that will provide specificity and sensitivity to diagnostics and improvement in prognostics. Investigations of atherosclerosis using direct proteomic studies have been challenged by the heterogeneity of the vascular tissue, hemodynamic forces as well as that of the plaque itself. An understanding of the effects of the two major mechanical forces, that is, cyclic stretch and shear stress, is indispensible for such investigations to fully comprehend their role in various clinically important signaling cascades inside the ECs. At present, our knowledge of the endothelial proteome under shear stress is still in its infancy (an extended list of proteins reported by proteomic investigations to be influenced by shear stress in ECs is provided as a SUPPLEMENTARY TABLE 1 [Supplementary material can be found online at www.informahealthcare.com/suppl/10.1586/ 14789450.2014.933673]). These studies chiefly highlighted upand downregulated proteins in response to applied shear stress conditions, but the role of these proteins in activation or inhibition of specific signaling pathways with regard to the response to shear stress needs further research. These studies have already identified some shear stress-responsive proteins that were not previously known and may pave the way for future investigations to understand the mechanisms linking cause and effect in cardiovascular diseases. Furthermore, the utilization of advanced proteomic tools in upcoming investigations of vascular cell–cell interactions as well as dissection of mechanisms through which hemodynamic forces interact with the vascular endothelium could help to explore still unraveled molecular mechanisms of vascular biology and pathophysiology. Five-year view
In vitro studies conducted so far to analyze the effect of shear stress on cellular responses leading to endothelial dysfunction and vascular pathologies have chiefly used experimental 615
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systems that allow exerting precisely quantifiable LSS on cultured layers of ECs. However, in vivo endothelial responses are sensitive to changes in flow characteristics. Moreover, there are multiple ways of deforming an EC by altering the flow profile, for example, at branches or curvatures of a blood vessel, that can hardly be mimicked by a cell culture model. Therefore, to further our understanding regarding the contribution of hemodynamic forces to cardiovascular complications, it is necessary to overcome the limitations of current experimental techniques to achieve an accurate representation of the in vivo conditions, improve the application of computational tools for accurate calculations of shear stress, utilize global as well as targeted proteomic approaches and investigate the individual and cumulative contributions of possible risk factors. Single cell culture models alone have limitations in their capacity to represent the precise clinical or pathophysiological disease entity which hinders understanding of the complexity of cardiovascular disorders. For instance, the development of atherosclerosis involves initial accumulation of blood-borne lipids underneath the thin layer of ECs followed by monocyte recruitment, transmigration and differentiation to macrophages in the subendothelial space where they scavenge lipids and enlarge into foam cells. The growth factors released by them attract smooth muscle cells to migrate from the media to the newly formed neointima further contributing to foam cells and early lesion formation in the vessel wall [104]. This complex phenomenon illustrates that for better representation of the underlying pathogenesis, namely, the altered hemodynamics in the context of endothelial dysfunction and predisposition to atherosclerotic lesions, at least coculture studies seem mandatory. Moreover, to investigate the role of shear stress in vascular mechanobiology by using in vitro models, development and improvement of methods
for preconditioning ECs to shear stress and then exposing them to additional changes in the local hemodynamics is a must. It will help to understand the preferential localization of atherosclerotic lesions to arterial branches and curvatures where blood flow and hence shear stress greatly deviates from its normal pattern in straight vessels. Proteomic analysis of ECs exposed to such experimental conditions may also unveil new paradigms for flow-dependent mechanotransduction inside ECs, namely, with regard to the biochemical pathways that are altered in response to changes in fluid mechanics with vital implications for EC functioning. Furthermore, the identification and characterization of new cellular components and signaling pathways by using established labeled and label-free quantitative proteomics procedures will help to better understand the disease mechanism and explore new therapeutic targets. It is likely that proteomic studies will dominate the identification of biomarkers associated with cardiovascular pathologies in the time to come. Acknowledgements
The authors acknowledge support by the German Research Foundation (DFG) and the Open Access Publication Fund of the University of Go¨ttingen. S Firasat is the recipient of a German Academic Exchange Service (DAAD) award. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues • Characteristic changes in blood flow are critical to vascular disease development and progression. • Endothelial cells (ECs) from different vascular sites are heterogeneous with respect to their physical and physiological characteristics. • Laminar shear stress and cyclic stretch generated by pulsatile blood flow are important mechanical forces that modulate endothelial function. • Loss of laminar shear stress is far more important than the relatively small increase in oscillatory shear stress to provoke a proatherosclerotic phenotype. • For implications of hemodynamic forces with reference to a particular cardiovascular complication a relevant laboratory model should be chosen. • Cell coculture studies are mandatory to accurately reflect the pathogenesis caused by altered hemodynamics, namely, in the context of endothelial dysfunction. • Differential proteome analysis of laminar and oscillating shear stress could provide further insights into conversion of functional phenotype of ECs to dysfunctional. • Improved in vitro devices to mimic pulsating fluid movement of the human vascular system are still wanted to explore endothelial physiology. • Proteomic investigations of vascular ECs in connection to clinical presentation are central to precisely detect novel disease and therapeutic biomarkers.
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Expert Rev. Proteomics 11(5), 611–619 (2014)
Sabika Firasat1,2, Markus Hecker3, Lutz Binder1 and Abdul R Asif*1 1 Institute of Clinical Chemistry UMG-Laboratories, Robert-Koch Str. 40, 37075 Goettingen, Germany 2 Department of Biosciences, University of Wah, Quaid Avenue, Wah Cantt, Pakistan 3 Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany *Author for correspondence: Tel.: +49 551 392 2945 Fax: +49 551 391 2505 [email protected]
The vascular endothelium lining the luminal surface of all blood vessels is constantly exposed to shear stress exerted by the flowing blood. Blood flow with high laminar shear stress confers protection by activation of antiatherogenic, antithrombotic and anti-inflammatory proteins, whereas low or oscillatory shear stress may promote endothelial dysfunction, thereby contributing to cardiovascular disease. Despite the usefulness of proteomic techniques in medical research, however, there are relatively few reports on proteome analysis of cultured vascular endothelial cells employing conditions that mimic in vivo shear stress attributes. This review focuses on the proteome studies that have utilized cultured endothelial cells to identify molecular mediators of shear stress and the roles they play in the regulation of endothelial function, and their ensuing effect on vascular function in general. It provides an overview on current strategies in shear stress-related proteomics and the key proteins mediating its effects which have been characterized so far. KEYWORDS: endothelial dysfunction • mass spectrometry • proteomics • shear stress
Vascular endothelium & shear stress
The endothelium lines the whole circulatory system from the heart to the smallest capillaries. However, endothelial cells (ECs) from different vascular sites are heterogeneous both in morphology and with respect to their protein expression and surface markers. This heterogeneity contributes to their physiological diversity across the vascular tree [1]. ECs regulate vascular homeostasis, tone and permeability; affect the balance between coagulation and fibrinolysis; contribute to inflammatory reactions; affect cellular propagation in the vessel wall; and interact with other cell types, for example, vascular smooth muscle cells, platelets, leukocytes, retinal pericytes, renal mesangial cells and macrophages through the production of different chemical mediators [2]. The inability of ECs to adequately carry out any of these functions is referred to as endothelial dysfunction, which is a hallmark of most forms of cardiovascular disease [3–5]. The endothelium is constantly exposed to hemodynamic forces due to pulsatile blood flow and pressure through the vasculature [6]. These hemodynamic forces include hydrostatic pressure created by the hydrostatic forces of blood within the blood vessels, cyclic stretch produced by longitudinal forces acting on the cell during vasomotion and shear stress, which informahealthcare.com
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are transduced between the ECs by defined intercellular connections, and the dragging frictional force applied tangential to the apical membrane of ECs by the blood flow [7]. Among these, cyclic stretch and shear stress are fundamental to modulating endothelial physiology, yet both make rather distinct and differing contributions to vascular disease [8–11]. Shear stress is of primary importance since it elicits a biochemical signal response in ECs [7,12]. The EC surface harbors numerous potential mechanoreceptors, for example, integrins, ion channels, receptor tyrosine kinases (such as vascular endothelial growth factor receptor, heterotrimeric G proteins and platelet-EC adhesion molecule-1 as well as supramolecular structures (or some of their constituents) such as the primary cilia, the apical glycocalyx, cell–cell junctions and the cytoskeleton, which, together with fluidity changes in the membrane lipid bilayer, aid in sensing or transmitting the initial biomechanical stimulus. These surface receptors or structures act as transducers of the flow-induced shear stress in ECs and trigger a complex network of interconnected intracellular pathways leading to adaptive changes in gene expression, cell metabolism and cell morphology [13–22]. Fluid shear stress is not uniform across the vascular system. The magnitude of shear stress ranges from 1 to 6 dynes/cm2 in the venous
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Table 1. Summary of proteomics studies on endothelial cells subjected to shear stress in vitro.
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Shear stress
Ref.
Type
Magnitude (dynes/cm2)
Exposure time
Proteomics strategy
Apparatus
In vitro model
Laminar
12–15
1 min, 2.5 min, 10 min
IMAC, SPE-CE-MS/MS
Cone and plate viscometer
BAECs
[58]
Oscillatory
0 ± 3.0
4h
LC–MS/MS
Dynamic flow system
BAECs
[93]
Laminar
15
10 min, 3 h, 6 h
ICAT, LC–MS/MS
Flow loop perfusion apparatus
BAECs
[50]
Laminar
10
18 h
ICAT, LC–MS/MS
Cone and plate viscometer
RVECs
[70]
Pulsatile oscillatory
23 0.02 ± 3
–
LC–MS/MS
Dynamic flow system
BAECs, HAECs
[82]
Laminar
25
30 min
DIGE, LC–MS/MS
Parallel plate flow chamber
HUVECs
[63]
Laminar
15
10 min, 3 h, 6 h
iTRAQ, LC–MS/MS
Parallel plate flow chamber
BAECs
[68]
Laminar
30
24 h
2DE, LC–MS/MS
Cone and plate viscometer
HUVECs
[54]
Laminar
5 12
6 h, various
LC–MS/MS
Parallel plate flow chamber
BAECs, HEK293, HUVECs
[57]
Laminar Oscillatory
15 8
24 h
SILAC, LC–MS/MS
Cone and plate viscometer
HUVECs
Steady
18 h
ICAT, LC–MS/MS
Cone and plate viscometer
RVECs
[83]
Pulsatile
10 and 30 5–15
Pulsatile
12 ± 4
1 h, 2 h, 4 h, 8 h
LC–MS/MS (PTM)
Dynamic flow system
HUVECs
[84]
[100]
BAECs: Bovine aortic endothelial cells; DIGE: Difference gel electrophoresis; HAECs: Human aortic endothelial cells; HEK293: Human embryonic kidney 293 cells; HUVECs: Human umbilical vein endothelial cells; ICAT: Isotope-coded affinity tags; IMAC: Immobilized metal affinity chromatography; iTRAQ: Isobaric tags for relative and absolute quantitation; PTM: Posttranslational modification; RVECs: Rat vascular endothelial cells; SILAC: Stable isotope labeling by amino acids in cell culture; SPE-CE: Solid phase extraction capillary electrophoresis.
network and from 10 to 70 dynes/cm2 in the arterial segment of the vasculature [23–26]. Straight parts of the vasculature experience steady and high laminar shear stress (LSS), which protects from atherosclerotic lesions [6,27,28]. On the other hand, at branch points and curvatures of the arterial system, blood flow is disturbed and wall shear stress is low, nonuniform and irregularly distributed [26,29,30]. This low and oscillatory shear stress (OSS) can cause EC dysfunction by inadequately modulating normal physiological EC signaling and gene expression, possibly leading to atherosclerosis and/or thrombosis [6,31–33], if accompanied by other risk factors that act on the vasculature, such as genetic predisposition, biochemical factors or lifestyle [34–36]. Proteomics of endothelial cells exposed to laminar shear stress
Proteomes are intricate and dynamic entities that are still poorly explored. Recent advances within the field of proteomics, including both global and targeted protocols, have 612
stimulated a transition from conventional protein identification to functional investigations, which will provide invaluable insight into disease pathogenesis [37]. The application of proteomic tools in the clinical setting is continuously increasing. However, relatively few studies have been investigating the proteome of ECs cultured under shear stress. Traditional techniques mainly based on immunological detection methods have identified some of the shear stress-responsive proteins [38–49]. But these techniques can identify only one protein at a time and heavily depend on the availability and quality of specific antibodies, limiting their feasibility for comprehensive analyses [50]. Using mass spectrometry-based strategies, however, the up- or downregulation of a large numbers of proteins in response to shear stress can be examined to disclose the molecular mechanisms contributing to endothelial dysfunction. Proteomic studies on cultured ECs involving the analysis of human umbilical vein ECs and ECs from various model species under different forms of shear stress are summarized in TABLE 1. Expert Rev. Proteomics 11(5), (2014)
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Endothelial shear stress proteomics
Endothelial dysfunction as an early sign of atherosclerosis is attributed to a decrease in nitric oxide (NO) bioactivity and enhancement in oxygen free radical formation. NO plays a major role in maintaining cardiovascular homeostasis as it is a potent vasodilator, antioxidant and anti-inflammatory mediator. It is synthesized by endothelial nitric oxide synthase (eNOS), and the most important physiological stimulus for eNOS expression is shear stress. Failure to adequately respond to this trigger for upregulating the production of eNOS should thus severely affect endothelial function. In fact a point mutation in the 5´-flanking region of the eNOS gene, -786T!C, has been reported to render the gene insensitive to LSS suppressing its transcription. As a consequence, human ECs homozygous for the eNOS CC gene variant are irresponsive to shear stress leading to endothelial dysfunction [51–54]. Severely reduced NO production, however, could indirectly affect the expression of other proteins in CC genotype cells. Asif et al. therefore compared the protein profiles of human umbilical vein ECs carrying the wild type (TT) or the mutant (CC) single nucleotide polymorphism after exposure to shear stress, using 2D electrophoresis and LC–MS/MS strategy. A total of 14 proteins were detected to be differentially expressed, of which eight proteins were similarly regulated in response to shear stress in both cell types. Four proteins involved in NO-dependent endoplasmic reticulum stress response, however, were upregulated by LSS in cells carrying the TT genotype. In contrast, cells carrying the CC genotype showed a unique increase in the transcription factor Egr-1-mediated expression of manganese-containing superoxide dismutase (SOD-2) in response to LSS. SOD-2 plays a critical role in protecting cells from superoxide, a key mediator of oxidative stress within the mitochondria. This increased SOD-2 expression in the CC genotype ECs may account for the fact that individuals homozygous for the -786T!C single nucleotide polymorphism do not show an earlier onset of coronary artery disease as compared to those carrying the TT genotype, as it may result in an antiatherosclerotic phenotype of these cells despite reduced eNOS activity, which in the long run, though, poses a higher risk after all [54,55]. eNOS activity is regulated by phosphorylation/ dephosphorylation of serine (Ser) and threonine residues by protein kinases/phosphatases [56,57]. Shear stress not only affects eNOS expression but also indirectly enhances eNOS activity via phosphorylation. To investigate the role of AMP-activated protein kinase in eNOS activation and NO bioavailability in ECs subjected to LSS, Chen et al. employed a nano-LC–MS/ MS-based proteomics approach. The study reported an enhanced AMP-activated protein kinase-dependent phosphorylation of eNOS Ser-633 in response to LSS, which acts as a functional signaling event for NO production, further underlining the role of LSS as a key activator of eNOS [57]. Gallis et al. also reported increased phosphorylation of eNOS in response to increased LSS in cultured bovine aortic endothelial cells (BAECs). Using immobilized metal affinity chromatography followed by solid phase extraction capillary electrophoresis and MS/MS analysis, two specific LSS-dependent eNOS informahealthcare.com
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phosphorylation sites, Ser-116 and Ser-1179, were identified. Activation of phosphatidylinositol 3-kinase and protein kinase B was found to be involved in shear stress-dependent eNOS phosphorylation and stimulation [58]. The stimulation of eNOS results in an increased production of NO, which, in turn, can interact with susceptible cysteine residues, resulting in S-nitrosylation of proteins. S-nitrosylation is an important posttranslational modification that plays a role in the modulation of cardiovascular function via the regulation of mitochondrial metabolism, intracellular Ca2+ handling, protein trafficking and cellular defense against apoptosis and oxidative stress [59–61]. Huang et al. analyzed the changes in S-nitrosylation of reactive cysteine residues present in endothelial proteins after the application of 25 dynes/cm2 LSS by the CyDye switch method (a modified biotin-switch method involving selective reduction of S-nitrosothiol using copper ions and ascorbate followed by in-gel detection of modified proteins by fluorescent cyanine dyes [62]). Using 2D fluorescence difference gel electrophoresis followed by MS/MS analysis, 12 proteins with a significant rise in S-nitrosylation after shear stress were identified. In addition, the S-nitrosylated cysteine residues of tropomyosin and vimentin were located in the hydrophobic motif of these proteins, leading to the suggestion that increased S-nitrosylation of these two cytoskeletal proteins is important for the adaptation and remodeling of ECs in response to laminar flow conditions [63]. Increased generation of endogenous reactive oxygen species is reported to cause cellular dysfunction in a number of pathophysiological conditions. The hyperglycemia-induced overproduction of superoxide appears to be the initial mechanism by which diabetes enhances reactive oxygen species generation in ECs. Hyperglycemia is thus involved in the abnormal endothelial response to shear stress via oxidative stress resulting in the impairment of cellular functions, for example, through nonenzymatic protein glycation, protein kinase C activation and modification of transcription factors [64–68]. The LC–MS/MS analysis of protein expression of BAECs pretreated with high glucose and subjected to LSS versus controls cultured at normal glucose levels revealed a set of differentially expressed proteins involved in structural integrity, biosynthesis and metabolism, cell proliferation and apoptosis, signal transduction and protein degradation, indicating a role for hyperglycemia in the development of a shear stress-mediated atheroprone phenotype of ECs [68]. These findings strongly indicate that shear stress is implicated in diabetes-related cardiovascular complications. To study the effect of different times of exposure to shear stress, Wang et al. established protein profiles of cultured BAECs by isotope-coded affinity tags labeling coupled with LC–MS/MS analysis. Out of a total of about 2000 proteins, 142, 213 and 186 proteins were regulated in response to 10 min, 3 h and 6 h of LSS, respectively. Among these were constituents of important signaling pathways such as integrins, G-protein-coupled receptors and glutamate receptors as well as elements of Notch, cAMP-mediated and PI3K/AKT pathways [50]. The PI3K/AKT pathway is involved in the activation 613
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of eNOS to produce high levels of NO in response to different stimuli, for example, growth factors, hormones [69] and shear stress. Using a labeled quantitative proteomics approach, Freed et al. analyzed the role of phosphatidylserine in the LSS-mediated protection of ECs against apoptosis [70]. Phosphatidylserine is known to activate protein kinases, which in turn control major signal transduction pathways of cell proliferation and cell survival [71,72]. They found that phosphatidylserine, which is present in the plasma membrane in ECs, is essential for the shear stress-mediated activation of the PI3K/ AKT survival pathway. These results further emphasize the role of shear stress in protecting ECs from apoptosis, which would be detrimental in atherosclerosis as it can lead to plaque rupture and thrombosis [73]. Fluid shear stress also contributes to the differentiation of ECs into a blood–brain barrier (BBB) phenotype that controls transport of biological substances required for brain metabolic processes and neuronal functions. The BBB consists of cerebral microvascular ECs characterized by the lack of fenestrations, few pinocytotic vesicles and the presence of well-developed tight junctions, which constitute a diffusion barrier to selectively exclude most blood-borne substances from entering the brain. In a proteome study using 2DE followed by densitometry analysis of a humanized dynamic in vitro BBB model, a significant upregulation of tight junction proteins in response to shear stress was shown. Thus, these observations highlight the role of LSS in promoting junctional integrity of ECs [74]. Besides its importance in neurodegenerative diseases [75,76], loss of endothelial cell–cell junction integrity at other sites of the vasculature, namely bifurcations and curvatures of large conduit arteries, is also associated with the development of atherosclerosis [77,78]. ECs in linear sections of the vasculature experience high LSS which is pulsatile in nature, with fluctuations in magnitude depending on the cardiac cycle [7]. In vitro studies often use a flow pattern that generates a temporally and spatially uniform, steady and positive shear stress. Nevertheless, there are some qualitative and quantitative differences in EC responses under pulsatile shear stress as compared to steady shear stress conditions [7,79–81]. In some recent studies, experimental set-ups were therefore modified to create pulsatile shear stress, for example, by utilizing dynamic flow systems [82–84]. Freed and Greene used a modified cone and plate viscometer to expose cultured rat vascular ECs to pulsatile shear stress to study the mechanisms associated with its prolonged defense of ECs against proapoptotic stimuli. The proteomic analysis of isolated TNF-a-associated signaling proteins using isotopic labeling quantitative proteomics revealed an increased expression of caspase recruitment domain-containing adaptor protein CARD9 in cells after treatment with steady and pulsatile shear flow prior to the exposure to apoptotic stimuli as compared to nontreated cells. In ECs, exposure to shear stress for 18 h conferred protection from TNF-a-induced apoptosis through an NO-independent mechanism that relied on de novo protein 614
synthesis [83]. CARD9 is a known activator of the NF-kB (nuclear factor kappa-light chain enhancer of activated B cells) pathway, which protects cells from apoptosis. These data suggest that laminar and pulsatile physiological shear stress contribute to the protection of ECs via NO production as well as NO-independent mechanisms. Pulsatile shear stress exerts antioxidative and anti-inflammatory effects on vascular ECs, in part by the induction of sirtuin 1 (SIRT1) [85]. SIRT1, a class III histone deacetylase, causes deacetylation of targets, for example, forkhead box O, peroxisome PPAR-g, PPAR-g coactivator 1a, NF-kB and eNOS [86,87]. To unveil the underlying mechanism involved in the flow-mediated upregulation of SIRT1, Wen et al. [84] identified phosphorylation sites based on nano-LC–MS/MS. Their study revealed that pulsatile shear stress induces an upregulation of SIRT1 level and activity by enhancing Ca2+/ calmodulin-dependent protein kinase kinase b (CaMKK-b) phosphorylation of SIRT1 at Ser-27 and Ser-47. The role of CaMKK-b in SIRT1 activation was further confirmed by the drastically increased atherosclerosis in mice lacking CaMKK-b or endothelial SIRT1 [84]. Proteomics of endothelial cells exposed to oscillatory shear stress
Shear stress experienced by ECs is not uniform throughout the vascular system, since it is a function of blood flow patterns across the vascular system produced by the cardiac cycle. In straight parts of vessels, blood flow exerts a high, directed LSS on ECs with fluctuations in magnitude depending on the phase of the cardiac cycle, whereas at branches and curvatures, laminar blood flow is disrupted resulting in separated flow patterns that generate a low, nonuniform OSS. At such locations, it is probably the lack of LSS as compared to the relatively moderate rise in OSS that is responsible for the observed change in phenotype and gene expression [7]. In addition, due to the reflection of the pulse wave at the bifurcation, ECs located at the outer vessel walls become cyclically stretched. This may be a factor that is equally important as the sharp drop in LSS promotes this phenotypic shift. As a consequence of the endothelial increase in proinflammatory gene expression, branches and curvatures of large conduit arteries are particularly prone to develop atherosclerotic lesions (atherosclerosis predilection sites) [88–90], as was shown in normal pigs and transgenic mice [91,92]. Moreover, the disturbed flow adds to postsurgical neointimal hyperplasia, in-stent restenosis, vein bypass graft failure, transplant vasculopathy and aortic valve calcification. In the venous system, disturbed flow due to reflux, stasis and outflow obstruction causes venous inflammation and thrombosis leading to the development of chronic venous diseases [25]. It is well known that before menopause women seem to be better protected against the progression of atherosclerosis than men, and this most likely is related to the level of estrogen. A protein profile established by LC–MS/MS of BAECs Expert Rev. Proteomics 11(5), (2014)
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pretreated with 17b-estradiol (E2) followed by exposure to OSS [93] showed that treatment with the hormone resulted in the downregulation of OSS-mediated NADPH oxidase 4 expression and superoxide production as well as in the upregulation of eNOS expression and NO production. The extent of low-density lipoprotein (LDL) modifications was also decreased in the presence of physiological concentrations of E2 under static as well as OSS conditions. Moreover, the LC–MS/MS analyses of xanthine oxidase-mediated protein nitration of LDL revealed that pretreatment with E2 reversed nitration (posttranslational modification) at the tyrosine residue 2535 of the a-2 helix domain. These data suggest that the effect of E2 is mediated, in part, by the downregulation of NADPH oxidase 4 and the upregulation of eNOS, leading to a suppression of oxidant production, which may explain the proatherogenic impact of OSS on arterial bifurcations. Furthermore, this study may explain how the indirect antioxidant activity of E2 contributes to the aforementioned gender-specific cardioprotection [94,95]. Ai et al. [82] analyzed the pathophysiological significance of SOD-2 in response to pulsatile and OSS using LDL particles to assess protein nitration via peroxynitrite (ONOO-). The analysis of apolipoprotein B-100 (apoB-100), the protein component of LDL, by LC–MS/MS revealed that OSS increased whereas pulsatile shear stress significantly decreased the extent of LDL protein nitration in comparison with static controls. OSS is a potent stimulus for oxidative stress, whereas SOD-2 is an important dismutase of superoxide anions (O2-) derived from the respiratory chain acting on the mitochondrial matrix [82]. OSS favors ONOO- formation through alteration of the O2- to NO ratio, leading to protein-bound nitrotyrosine formation [96–98]. Nitrotyrosine is prominently present in atherosclerosis-prone, OSS-exposed regions of the vasculature where SOD-2 is diminished, indicating implications of spatial variations in SOD-2 expression by specific shear stress patterns in atherosclerosis and inflammation [82,96,99]. In the abovementioned proteome studies, the impacts of either LSS- or OSS-induced regulatory or damaging effect(s) on ECs are compared with the situation under static conditions. Burghoff and Schrader [100], however, analyzed the secretome of ECs under static and shear stress (both LSS and OSS) conditions using a quantitative proteomics approach by stable isotope labeling of amino acids in cell culture. A total of 240 secreted proteins were identified in all three secretomes combined (under static, LSS and OSS conditions). Of these, 101 were differentially regulated under shear stress, highlighting the impact of shear stress on the contribution of ECs to the regulation of vascular homeostasis [100]. A subset of eight proteins specific for LSS and one comprising five proteins specific for OSS were detected, emphasizing dissimilarities of laminar and oscillating flow conditions. Most in vitro experimental models mimicking in vivo flow conditions have been used to study cellular responses to LSS. Few studies applied modified flow systems to create low, oscillatory flow which is experienced informahealthcare.com
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by ECs present at atheroprone regions of the vasculature [82,93,100]. However, a disadvantage of these modified systems is that experiments cannot be performed in parallel unless multiple devices are available. Another drawback is that acute effects of shear stress on ECs cannot be investigated due to the technical difficulties of subjecting a large number of cells to defined fluid flows over prolonged periods of time under sterile conditions. Some recent studies have proposed the use of orbital shakers to correlate shear stress with cellular responses to study many cases simultaneously [101–103]. However, to our knowledge, no such analysis of the ECs proteome in response to orbital shear stress has been published so far. One reason could be that it is difficult to precisely calculate the (heterogeneous) shear stress exerted by the rotating fluid on the EC monolayer – an obstacle that is likely to be overcome by applying computational fluid dynamic solvers in the future [102,103]. Expert commentary
Proteomic studies represent the current frontier strategy to understand disease mechanisms and identify novel diseaseassociated markers that will provide specificity and sensitivity to diagnostics and improvement in prognostics. Investigations of atherosclerosis using direct proteomic studies have been challenged by the heterogeneity of the vascular tissue, hemodynamic forces as well as that of the plaque itself. An understanding of the effects of the two major mechanical forces, that is, cyclic stretch and shear stress, is indispensible for such investigations to fully comprehend their role in various clinically important signaling cascades inside the ECs. At present, our knowledge of the endothelial proteome under shear stress is still in its infancy (an extended list of proteins reported by proteomic investigations to be influenced by shear stress in ECs is provided as a SUPPLEMENTARY TABLE 1 [Supplementary material can be found online at www.informahealthcare.com/suppl/10.1586/ 14789450.2014.933673]). These studies chiefly highlighted upand downregulated proteins in response to applied shear stress conditions, but the role of these proteins in activation or inhibition of specific signaling pathways with regard to the response to shear stress needs further research. These studies have already identified some shear stress-responsive proteins that were not previously known and may pave the way for future investigations to understand the mechanisms linking cause and effect in cardiovascular diseases. Furthermore, the utilization of advanced proteomic tools in upcoming investigations of vascular cell–cell interactions as well as dissection of mechanisms through which hemodynamic forces interact with the vascular endothelium could help to explore still unraveled molecular mechanisms of vascular biology and pathophysiology. Five-year view
In vitro studies conducted so far to analyze the effect of shear stress on cellular responses leading to endothelial dysfunction and vascular pathologies have chiefly used experimental 615
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systems that allow exerting precisely quantifiable LSS on cultured layers of ECs. However, in vivo endothelial responses are sensitive to changes in flow characteristics. Moreover, there are multiple ways of deforming an EC by altering the flow profile, for example, at branches or curvatures of a blood vessel, that can hardly be mimicked by a cell culture model. Therefore, to further our understanding regarding the contribution of hemodynamic forces to cardiovascular complications, it is necessary to overcome the limitations of current experimental techniques to achieve an accurate representation of the in vivo conditions, improve the application of computational tools for accurate calculations of shear stress, utilize global as well as targeted proteomic approaches and investigate the individual and cumulative contributions of possible risk factors. Single cell culture models alone have limitations in their capacity to represent the precise clinical or pathophysiological disease entity which hinders understanding of the complexity of cardiovascular disorders. For instance, the development of atherosclerosis involves initial accumulation of blood-borne lipids underneath the thin layer of ECs followed by monocyte recruitment, transmigration and differentiation to macrophages in the subendothelial space where they scavenge lipids and enlarge into foam cells. The growth factors released by them attract smooth muscle cells to migrate from the media to the newly formed neointima further contributing to foam cells and early lesion formation in the vessel wall [104]. This complex phenomenon illustrates that for better representation of the underlying pathogenesis, namely, the altered hemodynamics in the context of endothelial dysfunction and predisposition to atherosclerotic lesions, at least coculture studies seem mandatory. Moreover, to investigate the role of shear stress in vascular mechanobiology by using in vitro models, development and improvement of methods
for preconditioning ECs to shear stress and then exposing them to additional changes in the local hemodynamics is a must. It will help to understand the preferential localization of atherosclerotic lesions to arterial branches and curvatures where blood flow and hence shear stress greatly deviates from its normal pattern in straight vessels. Proteomic analysis of ECs exposed to such experimental conditions may also unveil new paradigms for flow-dependent mechanotransduction inside ECs, namely, with regard to the biochemical pathways that are altered in response to changes in fluid mechanics with vital implications for EC functioning. Furthermore, the identification and characterization of new cellular components and signaling pathways by using established labeled and label-free quantitative proteomics procedures will help to better understand the disease mechanism and explore new therapeutic targets. It is likely that proteomic studies will dominate the identification of biomarkers associated with cardiovascular pathologies in the time to come. Acknowledgements
The authors acknowledge support by the German Research Foundation (DFG) and the Open Access Publication Fund of the University of Go¨ttingen. S Firasat is the recipient of a German Academic Exchange Service (DAAD) award. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues • Characteristic changes in blood flow are critical to vascular disease development and progression. • Endothelial cells (ECs) from different vascular sites are heterogeneous with respect to their physical and physiological characteristics. • Laminar shear stress and cyclic stretch generated by pulsatile blood flow are important mechanical forces that modulate endothelial function. • Loss of laminar shear stress is far more important than the relatively small increase in oscillatory shear stress to provoke a proatherosclerotic phenotype. • For implications of hemodynamic forces with reference to a particular cardiovascular complication a relevant laboratory model should be chosen. • Cell coculture studies are mandatory to accurately reflect the pathogenesis caused by altered hemodynamics, namely, in the context of endothelial dysfunction. • Differential proteome analysis of laminar and oscillating shear stress could provide further insights into conversion of functional phenotype of ECs to dysfunctional. • Improved in vitro devices to mimic pulsating fluid movement of the human vascular system are still wanted to explore endothelial physiology. • Proteomic investigations of vascular ECs in connection to clinical presentation are central to precisely detect novel disease and therapeutic biomarkers.
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