Semi-automated mitral valve morphometry and computational stress analysis using 3D ultrasound

In vivo human mitral valves (MV) were imaged using real-time 3D transesophageal echocardiography (rt-3DTEE), and volumetric images of the MV at mid-systole were analyzed by user-initialized segmentation and 3D deformable modeling with continuous medial representation, a compact representation of shape. The resulting MV models were loaded with physiologic pressures using finite element analysis (FEA). We present the regional leaflet stress distributions predicted in normal and diseased (regurgitant) MVs. Rt-3DTEE, semi-automated leaflet segmentation, 3D deformable modeling, and FEA modeling of the in vivo human MV is tenable and useful for evaluation of MV pathology.     

Left ventricular endocardial longitudinal and transverse changes during isovolumic contraction and relaxation: a challenge

Left ventricular (LV) longitudinal and transverse geometric changes during isovolumic contraction and relaxation are still controversial. This confusion is compounded by traditional definitions of these phases of the cardiac cycle. High-resolution sonomicrometry studies might clarify these issues. Crystals were implanted in six sheep at the LV apex, fibrous trigones, lateral and posterior mitral annulus, base of the aortic right coronary sinus, anterior and septal endocardial wall, papillary muscle tips, and edge of the anterior and posterior mitral leaflets. Changes in distances were time related to LV and aortic pressures and to mitral valve opening. At the beginning of isovolumic contraction, while the mitral valve was still open, the LV endocardial transverse diameter started to shorten while the endocardial longitudinal diameter increased. During isovolumic relaxation, while the mitral valve was closed, LV transverse diameter started to increase while the longitudinal diameter continued to decrease. These findings are inconsistent with the classic definitions of the phases of the cardiac cycle.     

A three-dimensional insight into the complexity of flow convergence in mitral regurgitation: adjunctive benefit of anatomic regurgitant orifice area

Mitral effective regurgitant orifice area (EROA) using the flow convergence (FC) method is used to quantify the severity of mitral regurgitation (MR). However, it is challenging and prone to interobserver variability in complex valvular pathology. We hypothesized that real-time three-dimensional (3D) transesophageal echocardiography (RT3D TEE) derived anatomic regurgitant orifice area (AROA) can be a reasonable adjunct, irrespective of valvular geometry. Our goals were to 1) to determine the regurgitant orifice morphology and distance suitable for FC measurement using 3D computational flow dynamics and finite element analysis (FEA), and (2) to measure AROA from RT3D TEE and compare it with 2D FC derived EROA measurements. We studied 61 patients. EROA was calculated from 2D TEE images using the 2D-FC technique, and AROA was obtained from zoomed RT3DE TEE acquisitions using prototype software. 3D computational fluid dynamics by FEA were applied to 3D TEE images to determine the effects of mitral valve (MV) orifice geometry on FC pattern. 3D FEA analysis revealed that a central regurgitant orifice is suitable for FC measurements at an optimal distance from the orifice but complex MV orifice resulting in eccentric jets yielded nonaxisymmetric isovelocity contours close to the orifice where the assumptions underlying FC are problematic. EROA and AROA measurements correlated well (r = 0.81) with a nonsignificant bias. However, in patients with eccentric MR, the bias was larger than in central MR. Intermeasurement variability was higher for the 2D FC technique than for RT3DE-based measurements. With its superior reproducibility, 3D analysis of the AROA is a useful alternative to quantify MR when 2D FC measurements are challenging.     

Sensitivity of stress and strain calculations to passive material parameters in cardiac mechanical models using unloaded geometries

Cardiac stress (load) and strain (stretch) are widely studied indicators of cardiac function and outcome, but are difficult or impossible to directly measure in relation to the cardiac microstructure. An alternative approach is to estimate these states using computer methods and image-based measurements, but this still requires knowledge of the tissue material properties and the unloaded state, both of which are difficult to determine. In this work, we tested the sensitivity of these two interdependent unknowns (reference geometry and material parameters) on stress and strain calculations in cardiac tissue. Our study used a finite element model of the human ventricle, with a hyperelastic passive material model, and was driven by a cell model mediated active contraction. We evaluated 21 different published parameter sets for the five parameters of the passive material model, and for each set we optimised the corresponding unloaded geometry and contractility parameter to model a single pressure-volume loop. The resulting mechanics were compared, and calculated systolic stresses were largely insensitive to the chosen parameter set when an unloading algorithm was used. Meanwhile, material strain calculations varied substantially depending on the choice of material parameters. These results indicate that determining the correct material and unloaded configuration may be highly important to understand strain driven processes, but less so for calculating stress estimates.     

Isolated effect of geometry on mitral valve function for in silico model development

Computational models for the heart's mitral valve (MV) exhibit several uncertainties that may be reduced by further developing these models using ground-truth data-sets. This study generated a ground-truth data-set by quantifying the effects of isolated mitral annular flattening, symmetric annular dilatation, symmetric papillary muscle (PM) displacement and asymmetric PM displacement on leaflet coaptation, mitral regurgitation (MR) and anterior leaflet strain. MVs were mounted in an in vitro left heart simulator and tested under pulsatile haemodynamics. Mitral leaflet coaptation length, coaptation depth, tenting area, MR volume, MR jet direction and anterior leaflet strain in the radial and circumferential directions were successfully quantified at increasing levels of geometric distortion. From these data, increase in the levels of isolated PM displacement resulted in the greatest mean change in coaptation depth (70% increase), tenting area (150% increase) and radial leaflet strain (37% increase) while annular dilatation resulted in the largest mean change in coaptation length (50% decrease) and regurgitation volume (134% increase). Regurgitant jets were centrally located for symmetric annular dilatation and symmetric PM displacement. Asymmetric PM displacement resulted in asymmetrically directed jets. Peak changes in anterior leaflet strain in the circumferential direction were smaller and exhibited non-significant differences across the tested conditions. When used together, this ground-truth data-set may be used to parametrically evaluate and develop modelling assumptions for both the MV leaflets and subvalvular apparatus. This novel data may improve MV computational models and provide a platform for the development of future surgical planning tools.     

Microvesicles Derived from Adult Human Bone Marrow and Tissue Specific Mesenchymal Stem Cells Shuttle Selected Pattern of miRNAs

Background

Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).

Methodology/Principal Findings

MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation.

Conclusions

This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.     

Biphasic tissue Doppler waveforms during isovolumic phases are associated with asynchronous deformation of subendocardial and subepicardial layers.

Subendocardial and subepicardial layers of the left ventricle (LV) are characterized with right- and left-handed helical orientations of myocardial fibers. We investigated the origin of biphasic deformations of the LV wall during isovolumic contraction (IVC) and relaxation (IVR). In eight open-chest adult pigs, strain rates were measured along the right- and left-handed helical directions in the LV anterior wall by implanting 16 sonomicrometry crystals. Sonomicrometry strain rates were compared with the longitudinal subendocardial strain rates obtained by tissue Doppler imaging. During ejection and diastolic filling, shortening and lengthening occurred synchronously along the right- and left-handed helical directions. However, during IVC and IVR, the deformations were dissimilar in the two directions. Transmural shortening during IVC occurred along the right-handed helical direction and was accompanied with transient lengthening in the left-handed helical direction. Conversely, during IVR, the LV lengthened along the left-handed helical direction and shortened in the right-handed helical direction. Peak subendocardial strain rates obtained by tissue Doppler imaging during IVC and IVR correlated with corresponding sonomicrometry strain rate values obtained along the right- and left-handed helical directions (r = 0.81, P < 0.001 and r = 0.70, P = 0.001, respectively). Our data suggest that brief counterdirectional movements occur within the LV wall during IVC and IVR. Shortening along the right-handed helical direction is accompanied with reciprocal lengthening in the left-handed helical direction during IVC and vice versa during IVR. The results support an association between asynchronous deformation of subendocardial and subepicardial muscle fibers and the biphasic isovolumic movements observed with high-resolution tissue Doppler imaging.     

Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-alpha, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4(+) T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4(+) T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.     

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-β were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.     

Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury

Several studies demonstrated that treatment with mesenchymal stem cells (MSCs) reduces cisplatin mortality in mice. Microvesicles (MVs) released from MSCs were previously shown to favor renal repair in non lethal toxic and ischemic acute renal injury (AKI). In the present study we investigated the effects of MSC-derived MVs in SCID mice survival in lethal cisplatin-induced AKI. Moreover, we evaluated in vitro the effect of MVs on cisplatin-induced apoptosis of human renal tubular epithelial cells and the molecular mechanisms involved. Two different regimens of MV injection were used. The single administration of MVs ameliorated renal function and morphology, and improved survival but did not prevent chronic tubular injury and persistent increase in BUN and creatinine. Multiple injections of MVs further decreased mortality and at day 21 surviving mice showed normal histology and renal function. The mechanism of protection was mainly ascribed to an anti-apoptotic effect of MVs. In vitro studies demonstrated that MVs up-regulated in cisplatin-treated human tubular epithelial cells anti-apoptotic genes, such as Bcl-xL, Bcl2 and BIRC8 and down-regulated genes that have a central role in the execution-phase of cell apoptosis such as Casp1, Casp8 and LTA. In conclusion, MVs released from MSCs were found to exert a pro-survival effect on renal cells in vitro and in vivo, suggesting that MVs may contribute to renal protection conferred by MSCs.     

Biogenesis of bacterial membrane vesicles

Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram-negative bacteria both in vitro and in vivo . Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein–peptidoglycan (OM–PG) and OM protein–inner membrane protein (OM–PG–IM) interactions within the envelope structure. Modulation of OM–PG and OM–PG–IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM–PG and OM–PG–IM associations within the envelope structure, thus releasing OM as MVs.     

Material properties of the ovine mitral valve anterior leaflet in vivo from inverse finite element analysis

We measured leaflet displacements and used inverse finite-element analysis to define, for the first time, the material properties of mitral valve (MV) leaflets in vivo. Sixteen miniature radiopaque markers were sewn to the MV annulus, 16 to the anterior MV leaflet, and 1 on each papillary muscle tip in 17 sheep. Four-dimensional coordinates were obtained from biplane videofluoroscopic marker images (60 frames/s) during three complete cardiac cycles. A finite-element model of the anterior MV leaflet was developed using marker coordinates at the end of isovolumic relaxation (IVR; when the pressure difference across the valve is approximately 0), as the minimum stress reference state. Leaflet displacements were simulated during IVR using measured left ventricular and atrial pressures. The leaflet shear modulus (G(circ-rad)) and elastic moduli in both the commisure-commisure (E(circ)) and radial (E(rad)) directions were obtained using the method of feasible directions to minimize the difference between simulated and measured displacements. Group mean (+/-SD) values (17 animals, 3 heartbeats each, i.e., 51 cardiac cycles) were as follows: G(circ-rad) = 121 +/- 22 N/mm2, E(circ) = 43 +/- 18 N/mm2, and E(rad) = 11 +/- 3 N/mm2 (E(circ) > E(rad), P < 0.01). These values, much greater than those previously reported from in vitro studies, may result from activated neurally controlled contractile tissue within the leaflet that is inactive in excised tissues. This could have important implications, not only to our understanding of mitral valve physiology in the beating heart but for providing additional information to aid the development of more durable tissue-engineered bioprosthetic valves.     

Personalized Computational Modeling of Mitral Valve Prolapse: Virtual Leaflet Resection

Posterior leaflet prolapse following chordal elongation or rupture is one of the primary valvular diseases in patients with degenerative mitral valves (MVs). Quadrangular resection followed by ring annuloplasty is a reliable and reproducible surgical repair technique for treatment of posterior leaflet prolapse. Virtual MV repair simulation of leaflet resection in association with patient-specific 3D echocardiographic data can provide quantitative biomechanical and physiologic characteristics of pre- and post-resection MV function. We have developed a solid personalized computational simulation protocol to perform virtual MV repair using standard clinical guidelines of posterior leaflet resection with annuloplasty ring implantation. A virtual MV model was created using 3D echocardiographic data of a patient with posterior chordal rupture and severe mitral regurgitation. A quadrangle-shaped leaflet portion in the prolapsed posterior leaflet was removed, and virtual plication and suturing were performed. An annuloplasty ring of proper size was reconstructed and virtual ring annuloplasty was performed by superimposing the ring and the mitral annulus. Following the quadrangular resection and ring annuloplasty simulations, patient-specific annular motion and physiologic transvalvular pressure gradient were implemented and dynamic finite element simulation of MV function was performed. The pre-resection MV demonstrated a substantial lack of leaflet coaptation which directly correlated with the severe mitral regurgitation. Excessive stress concentration was found along the free marginal edge of the posterior leaflet involving the chordal rupture. Following the virtual resection and ring annuloplasty, the severity of the posterior leaflet prolapse markedly decreased. Excessive stress concentration disappeared over both anterior and posterior leaflets, and complete leaflet coaptation was effectively restored. This novel personalized virtual MV repair strategy has great potential to help with preoperative selection of the patient-specific optimal MV repair techniques, allow innovative surgical planning to expect improved efficacy of MV repair with more predictable outcomes, and ultimately provide more effective medical care for the patient.     

Modeling Active Contraction and Relaxation of Left Ventricle Using Different Zero-load Diastole and Systole Geometries for Better Material Parameter Estimation and Stress/Strain Calculations

Modeling ventricle active contraction based on in vivo data is extremely challenging because of complex ventricle geometry, dynamic heart motion and active contraction where the reference geometry (zero-stress geometry) changes constantly. A new modeling approach using different diastole and systole zero-load geometries was introduced to handle the changing zero-load geometries for more accurate stress/strain calculations. Echo image data were acquired from 5 patients with infarction (Infarct Group) and 10 without (Non-Infarcted Group). Echo-based computational two-layer left ventricle models using one zero-load geometry (1G) and two zero-load geometries (2G) were constructed. Material parameter values in Mooney-Rivlin models were adjusted to match volume data. Effective Young’s moduli (YM) were calculated for easy comparison. For diastole phase, begin-filling (BF) mean YM value in the fiber direction (YMf) was 738%higher than its end-diastole (ED) value (645.39 kPa vs. 76.97 kPa, p=3.38E-06). For systole phase, end-systole (ES) YMf was 903% higher than its begin-ejection (BE) value (1025.10 kPa vs. 102.11 kPa, p=6.10E-05). Comparing systolic and diastolic material properties, ES YMf was 59% higher than its BF value (1025.10 kPa vs. 645.39 kPa. p=0.0002). BE mean stress value was 514% higher than its ED value (299.69 kPa vs. 48.81 kPa, p=3.39E-06), while BE mean strain value was 31.5% higher than its ED value (0.9417 vs. 0.7162, p=0.004). Similarly, ES mean stress value was 562% higher than its BF value (19.74 kPa vs. 2.98 kPa, p=6.22E-05), and ES mean strain value was 264% higher than its BF value (0.1985 vs. 0.0546, p=3.42E-06). 2G models improved over 1G model limitations and may provide better material parameter estimation and stress/strain calculations.     

Virtual anthropology meets biomechanics

A meeting in Vienna in October 2010 brought together researchers using Virtual Anthropology (VA) and Finite Element Analysis (FEA) in order to explore the benefits and problems facing a collaboration between the two fields. FEA is used to test mechanical hypotheses in functional anatomy and VA complements and augments this process by virtue of its tools for acquiring data, for segmenting and preparing virtual specimens, and for generating reconstructions and artificial forms. This represents a critical methodological advance because geometry is one of the crucial inputs of FEA and is often the variable of interest in functional anatomy. However, we currently lack tools that quantitatively relate differences in geometry to differences in stress and strain, or that evaluate the impact on FEA of variation within and between biological samples. Thus, when comparing models of different geometry, we do not currently obtain sufficiently informative answers to questions such as “How different are these models, and in what manner are they different? Are they different in some anatomical regions but not others?" New methodologies must be developed in order to maximize the potential of FEA to address questions in comparative and evolutionary biology. In this paper we review these and other important issues that were raised during our Vienna meeting.     

Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano‐LC–MS/MS following 1D SDS‐PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung‐enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.     

Glycine significantly enhances bacterial membrane vesicle production: a powerful approach for isolation of LPS-reduced membrane vesicles of probiotic Escherichia coli

Bacterial membrane vesicles (MVs) have attracted strong interest in recent years as novel nanoparticle delivery platforms. Glycine is known to induce morphological changes in the outer layer of bacteria. We report here that glycine dramatically facilitates MV production in a flagella-deficient mutant of the non-pathogenic probiotic Escherichia coli strain Nissle 1917. Supplementation of culture medium with 1.0% glycine induced cell deformation at the early exponential phase, eventually followed by quasi-lysis during the late exponential to stationary phase. Glycine supplementation also significantly increased the number of MVs with enlarged particle size and altered the protein profile with an increase in the inner membrane and cytoplasmic protein contents as compared to non-induced MVs. Of note, the endotoxin activity of glycine-induced MVs was approximately eightfold or sixfold lower than that of non-induced MVs when compared at equal protein or lipid concentrations respectively. Nevertheless, glycine-induced MVs efficiently induced both immune responses in a mouse macrophage-like cell line and adjuvanticity in an intranasal vaccine mouse model, comparable to those of non-induced MVs. We propose that the present method of inducing MV production with glycine can be used for emerging biotechnological applications of MVs that have immunomodulatory activities, while dramatically reducing the presence of endotoxins.