CD98hc is a target for brain delivery of biotherapeutics.

Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TVCD98hc). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATVCD98hc) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATVCD98hc demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATVCD98hc that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TVCD98hc as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.

Arterial blood stealing as a mechanism of negative BOLD response: From the steady-flow with nonlinear phase separation to a windkessel-based model.

Blood Oxygen Level Dependent (BOLD) signal indirectly characterizes neuronal activity by measuring hemodynamic and metabolic changes in the nearby microvasculature. A deeper understanding of how localized changes in electrical, metabolic and hemodynamic factors translate into a BOLD signal is crucial for the interpretation of functional brain imaging techniques. While positive BOLD responses (PBR) are widely considered to be linked with neuronal activation, the origins of negative BOLD responses (NBR) have remained largely unknown. As NBRs are sometimes observed in close proximity of regions with PBR, a blood "stealing" effect, i.e., redirection of blood from a passive periphery to the area with high neuronal activity, has been postulated. In this study, we used the Hagen-Poiseuille equation to model hemodynamics in an idealized microvascular network that account for the particulate nature of blood and nonlinearities arising from the red blood cell (RBC) distribution (i.e., the Fåhraeus, Fåhraeus-Lindqvist and the phase separation effects). Using this detailed model, we evaluate determinants driving this "stealing" effect in a microvascular network with geometric parameters within physiological ranges. Model simulations predict that during localized cerebral blood flow (CBF) increases due to neuronal activation-hyperemic response, blood from surrounding vessels is reallocated towards the activated region. This stealing effect depended on the resistance of the microvasculature and the uneven distribution of RBCs at vessel bifurcations. A parsimonious model consisting of two-connected windkessel regions sharing a supplying artery was proposed to simulate the stealing effect with a minimum number of parameters. Comparison with the detailed model showed that the parsimonious model can reproduce the observed response for hematocrit values within the physiological range for different species. Our novel parsimonious model promise to be of use for statistical inference (top-down analysis) from direct blood flow measurements (e.g., arterial spin labeling and laser Doppler/Speckle flowmetry), and when combined with theoretical models for oxygen extraction/diffusion will help account for some types of NBRs.

Unraveling ChR2-driven stochastic Ca2+ dynamics in astrocytes: A call for new interventional paradigms.

Optogenetic targeting of astrocytes provides a robust experimental model to differentially induce Ca2+ signals in astrocytes in vivo. However, a systematic study quantifying the response of optogenetically modified astrocytes to light is yet to be performed. Here, we propose a novel stochastic model of Ca2+ dynamics in astrocytes that incorporates a light sensitive component-channelrhodopsin 2 (ChR2). Utilizing this model, we investigated the effect of different light stimulation paradigms on cells expressing select variants of ChR2 (wild type, ChETA, and ChRET/TC). Results predict that depending on paradigm specification, astrocytes might undergo drastic changes in their basal Ca2+ level and spiking probability. Furthermore, we performed a global sensitivity analysis to assess the effect of variation in parameters pertinent to the shape of the ChR2 photocurrent on astrocytic Ca2+ dynamics. Results suggest that directing variants towards the first open state of the ChR2 photocycle (o1) enhances spiking activity in astrocytes during optical stimulation. Evaluation of the effect of Ca2+ buffering and coupling coefficient in a network of ChR2-expressing astrocytes demonstrated basal level elevations in the stimulated region and propagation of calcium activity to unstimulated cells. Buffering reduced the diffusion range of Ca2+ within the network, thereby limiting propagation and influencing the activity of astrocytes. Collectively, the framework presented in this study provides valuable information for the selection of light stimulation paradigms that elicit desired astrocytic activity using existing ChR2 constructs, as well as aids in the engineering of future application-oriented optogenetic variants.

Contractile pericytes determine the direction of blood flow at capillary junctions.

The essential function of the circulatory system is to continuously and efficiently supply the O2 and nutrients necessary to meet the metabolic demands of every cell in the body, a function in which vast capillary networks play a key role. Capillary networks serve an additional important function in the central nervous system: acting as a sensory network, they detect neuronal activity in the form of elevated extracellular K+ and initiate a retrograde, propagating, hyperpolarizing signal that dilates upstream arterioles to rapidly increase local blood flow. Yet, little is known about how blood entering this network is distributed on a branch-to-branch basis to reach specific neurons in need. Here, we demonstrate that capillary-enwrapping projections of junctional, contractile pericytes within a postarteriole transitional region differentially constrict to structurally and dynamically determine the morphology of capillary junctions and thereby regulate branch-specific blood flow. We further found that these contractile pericytes are capable of receiving propagating K+-induced hyperpolarizing signals propagating through the capillary network and dynamically channeling red blood cells toward the initiating signal. By controlling blood flow at junctions, contractile pericytes within a functionally distinct postarteriole transitional region maintain the efficiency and effectiveness of the capillary network, enabling optimal perfusion of the brain.

The capillary Kir channel as sensor and amplifier of neuronal signals: Modeling insights on K+-mediated neurovascular communication.

Neuronal activity leads to an increase in local cerebral blood flow (CBF) to allow adequate supply of oxygen and nutrients to active neurons, a process termed neurovascular coupling (NVC). We have previously shown that capillary endothelial cell (cEC) inwardly rectifying K+ (Kir) channels can sense neuronally evoked increases in interstitial K+ and induce rapid and robust dilations of upstream parenchymal arterioles, suggesting a key role of cECs in NVC. The requirements of this signal conduction remain elusive. Here, we utilize mathematical modeling to investigate how small outward currents in stimulated cECs can elicit physiologically relevant spread of vasodilatory signals within the highly interconnected brain microvascular network to increase local CBF. Our model shows that the Kir channel can act as an "on-off" switch in cECs to hyperpolarize the cell membrane as extracellular K+ increases. A local hyperpolarization can be amplified by the voltage-dependent activation of Kir in neighboring cECs. Sufficient Kir density enables robust amplification of the hyperpolarizing stimulus and produces responses that resemble action potentials in excitable cells. This Kir-mediated excitability can remain localized in the stimulated region or regeneratively propagate over significant distances in the microvascular network, thus dramatically increasing the efficacy of K+ for eliciting local hyperemia. Modeling results show how changes in cEC transmembrane current densities and gap junctional resistances can affect K+-mediated NVC and suggest a key role for Kir as a sensor of neuronal activity and an amplifier of retrograde electrical signaling in the cerebral vasculature.

Modeling the role of endoplasmic reticulum-mitochondria microdomains in calcium dynamics.

Upon inositol trisphosphate (IP3) stimulation of non-excitable cells, including vascular endothelial cells, calcium (Ca2+) shuttling between the endoplasmic reticulum (ER) and mitochondria, facilitated by complexes called Mitochondria-Associated ER Membranes (MAMs), is known to play an important role in the occurrence of cytosolic Ca2+ concentration ([Ca2+]Cyt) oscillations. A mathematical compartmental closed-cell model of Ca2+ dynamics was developed that accounts for ER-mitochondria Ca2+ microdomains as the µd compartment (besides the cytosol, ER and mitochondria), Ca2+ influx to/efflux from each compartment and Ca2+ buffering. Varying the distribution of functional receptors in MAMs vs. the rest of ER/mitochondrial membranes, a parameter called the channel connectivity coefficient (to the µd), allowed for generation of [Ca2+]Cytoscillations driven by distinct mechanisms at various levels of IP3 stimulation. Oscillations could be initiated by the transient opening of IP3 receptors facing either the cytosol or the µd, and subsequent refilling of the respective compartment by Ca2+ efflux from the ER and/or the mitochondria. Only under conditions where the µd became the oscillation-driving compartment, silencing the Mitochondrial Ca2+ Uniporter led to oscillation inhibition. Thus, the model predicts that alternative mechanisms can yield [Ca2+]Cyt oscillations in non-excitable cells, and, under certain conditions, the ER-mitochondria µd can play a regulatory role.

waveCSD: A method for estimating transmembrane currents originated from propagating neuronal activity in the neocortex: Application to study cortical spreading depression.

BACKGROUND: Recent years have witnessed an upsurge in the development of methods for estimating current source densities (CSDs) in the neocortical tissue from their recorded local field potential (LFP) reflections using microelectrode arrays. Among these, methods utilizing linear arrays work under the assumption that CSDs vary as a function of cortical depth; whereas they are constant in the tangential direction, infinitely or in a confined cylinder. This assumption is violated in the analysis of neuronal activity propagating along the neocortical sheet, e.g. propagation of alpha waves or cortical spreading depression. NEW METHOD: Here, we developed a novel mathematical method (waveCSD) for CSD analysis of LFPs associated with a planar wave of neocortical neuronal activity propagating at a constant velocity towards a linear probe. RESULTS: Results show that the algorithm is robust to the presence of noise in LFP data and uncertainties in knowledge of propagation velocity. Also, results show high level of accuracy of the method in a wide range of electrode resolutions. Using in vivo experimental recordings from the rat neocortex, we employed waveCSD to characterize transmembrane currents associated with cortical spreading depressions. COMPARISON WITH EXISTING METHOD(S): Simulation results indicate that waveCSD has a significantly higher reconstruction accuracy compared to the widely-used inverse CSD method (iCSD), and the regularized kernel CSD method (kCSD), in the analysis of CSDs originating from propagating neuronal activity. CONCLUSIONS: The waveCSD method provides a theoretical platform for estimation of transmembrane currents from their LFPs in experimental paradigms involving wave propagation.

The yin and yang of KV channels in cerebral small vessel pathologies.

Cerebral SVDs encompass a group of genetic and sporadic pathological processes leading to brain lesions, cognitive decline, and stroke. There is no specific treatment for SVDs, which progress silently for years before becoming clinically symptomatic. Here, we examine parallels in the functional defects of PAs in CADASIL, a monogenic form of SVD, and in response to SAH, a common type of hemorrhagic stroke that also targets the brain microvasculature. Both animal models exhibit dysregulation of the voltage-gated potassium channel, KV 1, in arteriolar myocytes, an impairment that compromises responses to vasoactive stimuli and impacts CBF autoregulation and local dilatory responses to neuronal activity (NVC). However, the extent to which this channelopathy-like defect ultimately contributes to these pathologies is unknown. Combining experimental data with computational modeling, we describe the role of KV 1 channels in the regulation of myocyte membrane potential at rest and during the modest increase in extracellular potassium associated with NVC. We conclude that PA resting membrane potential and myogenic tone depend strongly on KV 1.2/1.5 channel density, and that reciprocal changes in KV channel density in CADASIL and SAH produce opposite effects on extracellular potassium-mediated dilation during NVC.

A "sweet-spot" for fluid-induced oscillations in the conditioning of stem cell-based engineered heart valve tissues.

Fluid-induced shear stresses are involved in the development of cardiovascular tissues. In a tissue engineering framework, this stimulus has also been considered as a mechanical regulator of stem cell differentiation. We recently demonstrated that the fluid-oscillating effect in combination with a physiologically-relevant shear stress magnitude contributes to the formation of stem cell-derived de novo heart valve tissues. However, the range of oscillations necessary to induce favorable gene expression and engineered tissue formation is unknown. In this study, we took a computational approach to establish a range of oscillatory shear stresses that may optimize in vitro valvular tissue growth. Taking a biomimetic approach, three physiologically-relevant flow waveforms from the human: (i) aorta, (ii) pulmonary artery and (iii) superior vena cava were utilized to simulate pulsatile flow conditions within a bioreactor that housed 3 tissue specimens. Results were compared to non-physiological pulsatile flow (NPPF) and cyclic flexure-steady flow (Flex-Flow) conditions. The oscillatory shear index (OSI) was used to quantify the fluid-induced oscillations occurring on the specimen surfaces. The range of mean OSI under the physiological conditions investigated was found to be 0.18 ≤ OSI ≤ 0.23. On the other hand, NPPF and Flex-Flow environments yielded a mean OSI of 0.37 and 0.11 respectively, which were 46% higher and 45% lower than physiological conditions. Moreover, we subsequently conducted OSI-based human bone marrow stem cell (HBMSC) culture experiments which resulted in preferential valvular gene expression and phenotype (significant upregulation of BMP, KLF2A, CD31 and α-SMA using an OSI of 0.23 in comparison to a lower OSI of 0.10 or a higher OSI of 0.38; p < .05). These findings suggest that a distinct range or a "sweet-spot" for physiological OSI exists in the mechanical conditioning of tissue engineered heart valves grown from stem cell sources. We conclude that in vitro heart valve matrix development could be further enhanced by simultaneous exposure of the engineered tissues to physiologically-relevant magnitudes of both fluid-induced oscillations and shear stresses.

Inherent rhythm of smooth muscle cells in rat mesenteric arterioles: An eigensystem formulation.

On the basis of experimental data and mathematical equations in the literature, we remodel the ionic dynamics of smooth muscle cells (SMCs) as an eigensystem formulation, which is valid for investigating finite variations of variables from the equilibrium such as in common experimental operations. This algorithm provides an alternate viewpoint from frequency-domain analysis and enables one to probe functionalities of SMCs' rhythm by means of a resonance-related mechanism. Numerical results show three types of calcium oscillations of SMCs in mesenteric arterioles: spontaneous calcium oscillation, agonist-dependent calcium oscillation, and agonist-dependent calcium spike. For simple single and double SMCs, we demonstrate properties of synchronization among complex signals related to calcium oscillations, and show different correlation relations between calcium and voltage signals for various synchronization and resonance conditions. For practical cell clusters, our analyses indicate that the rhythm of SMCs could (1) benefit enhancements of signal communications among remote cells, (2) respond to a significant calcium peaking against transient stimulations for triggering globally oscillating modes, and (3) characterize the globally oscillating modes via frog-leap (non-molecular-diffusion) calcium waves across inhomogeneous SMCs.

Comparison of transplantation of bone marrow stromal cells (BMSC) and stem cell mobilization by granulocyte colony stimulating factor after traumatic brain injury in rat.

BACKGROUND: Recent clinical studies of treating traumatic brain injury (TBI) with autologous adult stem cells led us to compare effect of intravenous injection of bone marrow mesenchymal stem cells (BMSC) and bone marrow hematopoietic stem cell mobilization, induced by granulocyte colony stimulating factor (G-CSF), in rats with a cortical compact device. METHODS: Forty adult male Wistar rats were injured with controlled cortical impact device and divided randomly into four groups. The treatment groups were injected with 2 × 106 intravenous bone marrow stromal stem cell (n = 10) and also with subcutaneous G-CSF (n = 10) and sham-operation group (n = 10) received PBS and "bromodeoxyuridine (Brdu)" alone, i.p. All injections were performed 1 day after injury into the tail veins of rats. All cells were labeled with Brdu before injection into the tail veins of rats. Functional neurological evaluation of animals was performed before and after injury using modified neurological severity scores (mNSS). Animals were sacrificed 42 days after TBI and brain sections were stained by Brdu immunohistochemistry. RESULTS: Statistically, significant improvement in functional outcome was observed in treatment groups compared with control group (P<0.01). mNSS showed no significant difference between the BMSC and G-CSF-treated groups during the study period (end of the trial). Histological analyses showed that Brdu-labeled (MSC) were present in the lesion boundary zone at 42nd day in all injected animals. CONCLUSION: In our study, we found that administration of a bone marrow-stimulating factor (G-CSF) and BMSC in a TBI model provides functional benefits.