The influence of voxelotor on cerebral blood flow and oxygen extraction in pediatric sickle cell disease.

ABSTRACT: Voxelotor is an inhibitor of sickle hemoglobin polymerization that is used to treat sickle cell disease. Although voxelotor has been shown to improve anemia, the clinical benefit on the brain remains to be determined. This study quantified the cerebral hemodynamic effects of voxelotor in children with sickle cell anemia (SCA) using noninvasive diffuse optical spectroscopies. Specifically, frequency-domain near-infrared spectroscopy combined with diffuse correlation spectroscopy were used to noninvasively assess regional oxygen extraction fraction (OEF), cerebral blood volume, and an index of cerebral blood flow (CBFi). Estimates of CBFi were first validated against arterial spin-labeled magnetic resonance imaging (ASL-MRI) in 8 children with SCA aged 8 to 18 years. CBFi was significantly positively correlated with ASL-MRI-measured blood flow (R2 = 0.651; P = .015). Next, a single-center, open-label pilot study was completed in 8 children with SCA aged 4 to 17 years on voxelotor, monitored before treatment initiation and at 4, 8, and 12 weeks (NCT05018728). By 4 weeks, both OEF and CBFi significantly decreased, and these decreases persisted to 12 weeks (both P < .05). Decreases in CBFi were significantly correlated with increases in blood hemoglobin (Hb) concentration (P = .025), whereas the correlation between decreases in OEF and increases in Hb trended toward significance (P = .12). Given that previous work has shown that oxygen extraction and blood flow are elevated in pediatric SCA compared with controls, these results suggest that voxelotor may reduce cerebral hemodynamic impairments. This trial was registered at www.ClinicalTrials.gov as #NCT05018728.

EGFR activation attenuates the mechanical threshold for integrin tension and focal adhesion formation.

Mechanical forces, growth factors and the extracellular matrix all play crucial roles in cell adhesion. To understand how epidermal growth factor receptor (EGFR) impacts the mechanics of adhesion, we employed tension gauge tether (TGT) probes displaying the integrin ligand cRGDfK and quantified integrin tension. EGF exposure significantly increased spread area, cell circularity, integrated integrin tension, mechanical rupture density, radial organization and size of focal adhesions in Cos-7 cells on TGT surfaces. These findings suggest that EGFR regulates integrin tension and the spatial organization of focal adhesions. Additionally, we found that the mechanical tension threshold for outside-in integrin activation is tunable by EGFR. Parallel genetic and pharmacologic strategies demonstrated that these phenotypes are driven by ligand-dependent EGFR signaling. Our results establish a novel mechanism whereby EGFR regulates integrin activation and cell adhesion, providing control over cellular responses to the environment.This article has an associated First Person interview with the first author of the paper.

Desmoglein 3 Order and Dynamics in Desmosomes Determined by Fluorescence Polarization Microscopy.

Desmosomes are macromolecular cell-cell junctions that provide adhesive strength in epithelial tissue. Desmosome function is inseparably linked to structure, and it is hypothesized that the arrangement, or order, of desmosomal cadherins in the intercellular space is critical for adhesive strength. However, due to desmosome size, molecular complexity, and dynamics, the role that order plays in adhesion is challenging to study. Herein, we present an excitation resolved fluorescence polarization microscopy approach to measure the spatiotemporal dynamics of order and disorder of the desmosomal cadherin desmoglein 3 (Dsg3) in living cells. Simulations were used to establish order factor as a robust metric for quantifying the spatiotemporal dynamics of order and disorder. Order factor measurements in keratinocytes showed the Dsg3 extracellular domain is ordered at the individual desmosome, single cell, and cell population levels compared to a series of disordered controls. Desmosomal adhesion is Ca2+ dependent, and reduction of extracellular Ca2+ leads to a loss of adhesion measured by dispase fragmentation assay (λ = 15.1 min). Live cell imaging revealed Dsg3 order decreased more rapidly (λ = 5.5 min), indicating that cadherin order is not required for adhesion. Our results suggest that rapid disordering of cadherins can communicate a change in extracellular Ca2+ concentration to the cell, leading to a downstream loss of adhesion. Fluorescence polarization is an effective bridge between protein structure and complex dynamics and the approach presented here is broadly applicable to studying order in macromolecular structures.

Normative cerebral microvascular blood flow waveform morphology assessed with diffuse correlation spectroscopy.

Microvascular cerebral blood flow exhibits pulsatility at the cardiac frequency that carries valuable information about cerebrovascular health. This study used diffuse correlation spectroscopy to quantify normative features of these waveforms in a cohort of thirty healthy adults. We demonstrate they are sensitive to changes in vascular tone, as indicated by pronounced morphological changes with hypercapnia. Further, we observe significant sex-based differences in waveform morphology, with females exhibiting higher flow, greater area-under-the-curve, and lower pulsatility. Finally, we quantify normative values for cerebral critical closing pressure, i.e., the minimum pressure required to maintain flow in a given vascular region.

Three-dimensional light-field microendoscopy with a GRIN lens array.

Optical endoscopy has emerged as an indispensable clinical tool for modern minimally invasive surgery. Most systems primarily capture a 2D projection of the 3D surgical field. Currently available 3D endoscopes can restore stereoscopic vision directly by projecting laterally shifted views of the operating field to each eye through 3D glasses. These tools provide surgeons with informative 3D visualizations, but they do not enable quantitative volumetric rendering of tissue. Therefore, advanced tools are desired to quantify tissue tomography for high precision microsurgery or medical robotics. Light-field imaging suggests itself as a promising solution to the challenge. The approach can capture both the spatial and angular information of optical signals, permitting the computational synthesis of the 3D volume of an object. In this work, we present GRIN lens array microendoscopy (GLAM), a single-shot, full-color, and quantitative 3D microendoscopy system. GLAM contains integrated fiber optics for illumination and a GRIN lens array to capture the reflected light field. The system exhibits a 3D resolution of ∼100 µm over an imaging depth of ∼22 mm and field of view up to 1 cm2. GLAM maintains a small form factor consistent with the clinically desirable design, making the system readily translatable to a clinical prototype.

Protein exchange is reduced in calcium-independent epithelial junctions.

Desmosomes are cell-cell junctions that provide mechanical integrity to epithelial and cardiac tissues. Desmosomes have two distinct adhesive states, calcium-dependent and hyperadhesive, which balance tissue plasticity and strength. A highly ordered array of cadherins in the adhesive interface is hypothesized to drive hyperadhesion, but how desmosome structure confers adhesive state is still elusive. We employed fluorescence polarization microscopy to show that cadherin order is not required for hyperadhesion induced by pharmacologic and genetic approaches. FRAP experiments in cells treated with the PKCα inhibitor Gö6976 revealed that cadherins, plakoglobin, and desmoplakin have significantly reduced exchange in and out of hyperadhesive desmosomes. To test whether this was a result of enhanced keratin association, we used the desmoplakin mutant S2849G, which conferred reduced protein exchange. We propose that inside-out regulation of protein exchange modulates adhesive function, whereby proteins are "locked in" to hyperadhesive desmosomes while protein exchange confers plasticity on calcium-dependent desmosomes, thereby providing rapid control of adhesion.

Bridging the gap: Super-resolution microscopy of epithelial cell junctions.

Cell junctions are critical for cell adhesion and communication in epithelial tissues. It is evident that the cellular distribution, size, and architecture of cell junctions play a vital role in regulating function. These details of junction architecture have been challenging to elucidate in part due to the complexity and size of cell junctions. A major challenge in understanding these features is attaining high resolution spatial information with molecular specificity. Fluorescence microscopy allows localization of specific proteins to junctions, but with a resolution on the same scale as junction size, rendering internal protein organization unobtainable. Super-resolution microscopy provides a bridge between fluorescence microscopy and nanoscale approaches, utilizing fluorescent tags to reveal protein organization below the resolution limit. Here we provide a brief introduction to super-resolution microscopy and discuss novel findings into the organization, structure and function of epithelial cell junctions.