A root cause analysis to identify the mechanistic drivers of immunogenicity against the anti-VEGF biotherapeutic brolucizumab.

Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.

Quantifying the relative contributions of different solute carriers to aggregate substrate transport.

Determining the contributions of different transporter species to overall cellular transport is fundamental for understanding the physiological regulation of solutes. We calculated the relative activities of Solute Carrier (SLC) transporters using the Michaelis-Menten equation and global fitting to estimate the normalized maximum transport rate for each transporter (Vmax). Data input were the normalized measured uptake of the essential neutral amino acid (AA) L-leucine (Leu) from concentration-dependence assays performed using Xenopus laevis oocytes. Our methodology was verified by calculating Leu and L-phenylalanine (Phe) data in the presence of competitive substrates and/or inhibitors. Among 9 potentially expressed endogenous X. laevis oocyte Leu transporter species, activities of only the uniporters SLC43A2/LAT4 (and/or SLC43A1/LAT3) and the sodium symporter SLC6A19/B0AT1 were required to account for total uptake. Furthermore, Leu and Phe uptake by heterologously expressed human SLC6A14/ATB0,+ and SLC43A2/LAT4 was accurately calculated. This versatile systems biology approach is useful for analyses where the kinetics of each active protein species can be represented by the Hill equation. Furthermore, its applicable even in the absence of protein expression data. It could potentially be applied, for example, to quantify drug transporter activities in target cells to improve specificity.

The Bohr Effect Is Not a Likely Promoter of Renal Preglomerular Oxygen Shunting.

The aim of this study was to evaluate whether possible preglomerular arterial-to-venous oxygen shunting is affected by the interaction between renal preglomerular carbon dioxide and oxygen transport. We hypothesized that a reverse (venous-to-arterial) shunting of carbon dioxide will increase partial pressure of carbon dioxide and decrease pH in the arteries and thereby lead to increased oxygen offloading and consequent oxygen shunting. To test this hypothesis, we employed a segment-wise three-dimensional computational model of coupled renal oxygen and carbon dioxide transport, wherein coupling is achieved by shifting the oxygen-hemoglobin dissociation curve in dependence of local changes in partial pressure of carbon dioxide and pH. The model suggests that primarily due to the high buffering capacity of blood, there is only marginally increased acidity in the preglomerular vasculature compared to systemic arterial blood caused by carbon dioxide shunting. Furthermore, effects of carbon dioxide transport do not promote but rather impair preglomerular oxygen shunting, as the increase in acidity is higher in the veins compared to that in the arteries. We conclude that while substantial arterial-to-venous oxygen shunting might take place in the postglomerular vasculature, the net amount of oxygen shunted at the preglomerular vasculature appears to be marginal.

Drug deposition in coronary arteries with overlapping drug-eluting stents.

Drug-eluting stents are accepted as mainstream endovascular therapy, yet concerns for their safety may be under-appreciated. While failure from restenosis has dropped to below 5%, the risk of stent thrombosis and associated mortality remain relatively high. Further optimization of drug release is required to minimize thrombosis risk while maintaining therapeutic dose. The complex three-dimensional geometry of deployed stents together with the combination of diffusive and advective drug transport render an intuitive understanding of the situation exceedingly difficult. In situations such as this, computational modeling has proven essential, helping define the limits of efficacy, determine the mode and mechanism of drug release, and identify alternatives to avoid toxicity. A particularly challenging conformation is encountered in coronary arteries with overlapping stents. To study hemodynamics and drug deposition in such vessels we combined high-resolution, multi-scale ex vivo computed tomography with a flow and mass transfer computational model. This approach ensures high geometric fidelity and precise, simultaneous calculation of blood flow velocity, shear stress and drug distribution. Our calculations show that drug uptake by the arterial tissue is dependent both on the patterns of flow disruption near the wall, as well as on the relative positioning of drug-eluting struts. Overlapping stent struts lead to localized peaks of drug concentration that may increase the risk of thrombosis. Such peaks could be avoided by anisotropic stent structure or asymmetric drug release designed to yield homogeneous drug distribution along the coronary artery and, at the least, suggest that these issues need to remain in the forefront of consideration in clinical practice.

Renal oxygenation: preglomerular vasculature is an unlikely contributor to renal oxygen shunting.

The primary aim of this study was to assess the plausibility of preglomerular arterial-to-venous oxygen shunting in the kidney. To this end, we have developed a segment-wise three-dimensional computational model that takes into account transport processes in arteries, veins, cortical tissue, and capillaries. Our model suggests that the amount of preglomerular oxygen shunting is negligible. Consequently, it is improbable that preglomerular shunting contributes to the hypothesized regulation of renal oxygenation. Cortical tissue oxygenation is more likely determined by the interplay between oxygen supply, either from the preglomerular vasculature or from capillaries, and oxygen consumption. We show that reported differences in permeability to oxygen between perfused and unperfused tissue may be explained by what we refer to as advection-facilitated diffusion. We further show that the preglomerular vasculature is the primary source of oxygen for the tissue when cortical consumption is high or renal arterial blood is highly oxygenated, i.e., under hyperoxemic conditions. Conversely, when oxygen demand in the tissue is decreased, or under hypoxemic conditions, oxygen is supplied predominantly by capillaries.

Choosing the optimal wall shear parameter for the prediction of plaque location-A patient-specific computational study in human left coronary arteries.

OBJECTIVE: While the correlation of atherosclerotic plaque locations with local wall shear stress magnitude has been evaluated previously by other investigators in both right (RCA) and left coronary arteries (LCA), the relative performance of average wall shear stress (AWSS), average wall shear stress gradient (AWSSG), oscillatory shear index (OSI) and relative residence time (RRT) as indicators of potential atherosclerotic plaque locations has not been studied for the LCA. Here we determine the performance of said wall shear parameters in the LCA for the prediction of plaque development locations and compare these results to those previously found in the RCA. METHODS: We obtained 30 patient-specific geometries (mean age 67.1 (± 9.2) years, all with stable angina) of the LCA using dual-source computed tomography and virtually removed any plaque present. We then performed computational fluid dynamics simulations to calculate the wall shear parameters. RESULTS: For the 96 total plaques, AWSS had a higher sensitivity for the prediction of plaque locations (86 ± 25%) than AWSSG (65 ± 37%, p<0.05), OSI (67 ± 32%, p<0.01) or RRT (48 ± 38%, p<0.001). RRT had a higher PPV (49 ± 36%) than AWSS (31 ± 20%, p<0.05) or AWSSG (16 ± 12%, p<0.001). Segment 5 of the LCA presented with overall low values for sensitivity and PPV. Parameter performance in the remainder of the LCA was comparable to that in the RCA. CONCLUSIONS: AWSS features remarkably high sensitivity, but does not reach the PPV of RRT. This may indicate that while low wall shear stress is necessary for plaque formation, its presence alone is not sufficient to predict future plaque locations. Time dependent factors have to be taken into account as well.

Computed high concentrations of low-density lipoprotein correlate with plaque locations in human coronary arteries.

Subendothelial accumulation of low-density lipoprotein (LDL) in arterial walls is an initiator of atherosclerotic plaque formation. We report here on the correlation between healthy state subendothelial LDL concentration distribution and sites of subsequent plaque formation in coronary arteries of patients with coronary artery disease (CAD). We acquired left (LCA) and right coronary artery (RCA) and atherosclerotic plaque geometries of 60 patients with CAD using dual-source computed tomography angiography. After virtually removing all plaques to obtain an approximation of the arteries' healthy state, we calculated LDL concentration in the artery walls as a function of local lumen-side shear stress. We found that maximum subendothelial LDL concentrations at plaque locations were, on average, 45% (RCA) and 187% (LCA) higher than the respective average subendothelial concentration. Our results demonstrate that locally elevated subendothelial LDL concentration correlates with subsequent plaque formation at the same location.

Choosing the optimal wall shear parameter for the prediction of plaque location-A patient-specific computational study in human right coronary arteries.

BACKGROUND: Average wall shear-stress (AWSS), average wall shear-stress gradient (AWSSG), oscillatory shear index (OSI) and relative residence time (RRT) are believed to predict areas vulnerable to plaque formation in the coronary arteries. Our aim was to analyze the correlation of these parameters in patients' vessels before the onset of atherosclerosis to the specific plaque sites thereafter, and to compare the parameters' sensitivity and positive predictive value. METHODS: We obtained 30 patient-specific geometries (mean age 67.1 (+ or - 9.2) years, all with stable angina) of the right coronary artery (RCA) using dual-source computed tomography (CT) and virtually removed any plaque present. We then performed computational fluid dynamics (CFD) simulations to calculate the wall shear parameters. RESULTS: For the 120 total plaques, AWSS had on average a higher sensitivity for the prediction of plaque locations (72 + or - 25%) than AWSSG (68 + or - 36%), OSI (60 + or - 30%, p<0.05), and RRT (69 + or - 59%); while OSI had a higher positive predict value (PPV) (68 + or - 34%) than AWSS (47 + or - 27%, p<0.001), AWSSG (37 + or - 23, p<0.001) and RRT (59 + or - 34%). A significant difference was also found between AWSSG and RRT (p<0.01) concerning PPV. CONCLUSIONS: OSI and RRT are the optimal parameters when the number of false positives is to be minimized. AWSS accurately identifies the largest number of plaques, but produces more false positives than OSI and RRT.

Patient-specific three-dimensional simulation of LDL accumulation in a human left coronary artery in its healthy and atherosclerotic states.

We calculate low-density lipoprotein (LDL) transport from blood into arterial walls in a three-dimensional, patient-specific model of a human left coronary artery. The in vivo anatomy data are obtained from computed tomography images of a patient with coronary artery disease. Models of the artery anatomy in its healthy and diseased states are derived after segmentation of the vessel lumen, with and without the detected plaque, respectively. Spatial shear stress distribution at the endothelium is determined through the reconstruction of the arterial blood flow field using computational fluid dynamics. The arterial endothelium is represented by a shear stress-dependent, three-pore model, taking into account blood plasma and LDL passage through normal junctions, leaky junctions, and the vesicular pathway. Intraluminal pressures of 70 and 120 mmHg are employed as the normal and hypertensive operating pressures, respectively. By applying our model to both the healthy and diseased states, we show that the location of the plaque in the diseased state corresponds to one of the two sites with predicted high-LDL concentration in the healthy state. We further show that, in the diseased state, the site with high-LDL concentration has shifted distal to the plaque, which is in agreement with the clinical observation that plaques generally grow in the downstream direction. We also demonstrate that hypertension leads to increased number of regions with high-LDL concentration, elucidating one of the ways in which hypertension may promote atherosclerosis.

CFD and PTV steady flow investigation in an anatomically accurate abdominal aortic aneurysm.

There is considerable interest in computational and experimental flow investigations within abdominal aortic aneurysms (AAAs). This task stipulates advanced grid generation techniques and cross-validation because of the anatomical complexity. The purpose of this study is to examine the feasibility of velocity measurements by particle tracking velocimetry (PTV) in realistic AAA models. Computed tomography and rapid prototyping were combined to digitize and construct a silicone replica of a patient-specific AAA. Three-dimensional velocity measurements were acquired using PTV under steady averaged resting boundary conditions. Computational fluid dynamics (CFD) simulations were subsequently carried out with identical boundary conditions. The computational grid was created by splitting the luminal volume into manifold and nonmanifold subsections. They were filled with tetrahedral and hexahedral elements, respectively. Grid independency was tested on three successively refined meshes. Velocity differences of about 1% in all three directions existed mainly within the AAA sack. Pressure revealed similar variations, with the sparser mesh predicting larger values. PTV velocity measurements were taken along the abdominal aorta and showed good agreement with the numerical data. The results within the aneurysm neck and sack showed average velocity variations of about 5% of the mean inlet velocity. The corresponding average differences increased for all velocity components downstream the iliac bifurcation to as much as 15%. The two domains differed slightly due to flow-induced forces acting on the silicone model. Velocity quantification through narrow branches was problematic due to decreased signal to noise ratio at the larger local velocities. Computational wall pressure and shear fields are also presented. The agreement between CFD simulations and the PTV experimental data was confirmed by three-dimensional velocity comparisons at several locations within the investigated AAA anatomy indicating the feasibility of this approach.

Identification of atherosclerotic lesion-prone sites through patient-specific simulation of low-density lipoprotein accumulation.

We present a patient-specific model of low-density lipoprotein (LDL) transport from blood into arterial walls. To this end, the arterial endothelium is represented by a shear-stress dependent three-pore model taking into account blood plasma and LDL passage through the vesicular pathway, normal junctions and leaky junctions. We virtually remove atherosclerotic plaque from an in-vivo left coronary artery computed tomography (CT) dataset to obtain an approximation of the artery anatomy in its healthy state. By applying our model, we show that the location of the plaque in the diseased state corresponds to one of the two sites with predicted high LDL concentration in the healthy state. We further show that in the diseased state, the site with high LDL concentration has shifted distally, which is in agreement with the clinical observation that plaques generally grow in downstream direction.

Computational modeling of coupled blood-wall mass transport of LDL: effects of local wall shear stress.

The work herein represents a novel approach for the modeling of low-density lipoprotein (LDL) transport from the artery lumen into the arterial wall, taking into account the effects of local wall shear stress (WSS) on the endothelial cell layer and its pathways of volume and solute flux. We have simulated LDL transport in an axisymmetric representation of a stenosed coronary artery, where the endothelium is represented by a three-pore model that takes into account the contributions of the vesicular pathway, normal junctions, and leaky junctions also employing the local WSS to yield the overall volume and solute flux. The fraction of leaky junctions is calculated as a function of the local WSS based on published experimental data and is used in conjunction with the pore theory to determine the transport properties of this pathway. We have found elevated levels of solute flux at low shear stress regions because of the presence of a larger number of leaky junctions compared with high shear stress regions. Accordingly, we were able to observe high LDL concentrations in the arterial wall in these low shear stress regions despite increased filtration velocity, indicating that the increase in filtration velocity is not sufficient for the convective removal of LDL.