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Activation of SCD RBC adhesion by oxidative stress
of adhesion. However, random precapillary obstruction by a small number of dense RBC also contributes to VOC as well as the entrapment of leukocytes and platelets.11-14
Sickle RBC are very heterogeneous in terms of age, shape and surface proteins. These variabilities account for the differences observed in cell adhesion and resistance to shear stress under flow conditions.11 In SCD, among other proteins and mechanisms, adhesion proteins LW/ICAM-4 (Landsteiner-Wiener/intercellular adhesion molecule-4) and Lu/BCAM (Lutheran/basal cell adhesion molecule) are abnormally activated and believed to prime adhesion of RBC to endothelial cells and/or subendothelial matrix pro- teins exposed to the bloodstream following vascular dam- age, contributing to microvasculature blockade.10,15-21
Lu/BCAM is an adhesion molecule with wide tissue dis- tribution.22,23 Lu/BCAM-mediated cell adhesion to laminin can be triggered either by the phosphorylation of its serine 62117,24 or by the dissociation of its cytoplasmic domain from the spectrin-based skeleton.25,26 In SCD, phosphory- lation of Lu/BCAM was shown to occur in low-density (LD) RBC,27 mainly reticulocytes, consistent with the adhesion of these cells to laminin.27,28 However, despite firm adhesion to laminin of high-density (HD) RBC, Lu/BCAM phosphorylation is very minor in this subpop- ulation and these cells do not respond to cAMP inducers such as forskolin.28 The mechanism underlying this increased adhesion is still unknown.
In this study, we investigated the molecular mechanism responsible for the increased adhesion of sickle HD RBC to laminin. We provide evidence for the implication of oxidative stress in post-translational modifications of Lu/BCAM that impact its distribution and cis-interactions at the cell surface and activate its adhesive function.
Methods
Patients
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Committee (n°3215 CPP Ile de France III). Blood samples were recovered from blood tubes drawn for medical care at Necker Hospital (Paris) after written informed consent. Blood samples were collected on ethyl- enediaminetetraacetic acid (EDTA) from a total of 39 patients with sickle cell anemia (SS and Sβ° genotypes) (females and males; median age: 8 years [Min-Max, 2-53 years]), and from 26 healthy donors (age range, 18-70, as per Etablissement Français du Sang [EFS] criteria). Sickle patients were not on a regular transfusion program nor under hydroxyurea (HU) treatment. All experiments were performed with fresh blood samples, within 2 hours after blood was drawn.
Percoll fractionation
RBC subpopulations were obtained from sickle whole blood fractionation as previously described,27 using a Percoll triple-densi- ty fractionation (densities: 1.076, 1.096, and 1.11). Three different density layers were obtained and collected as follows: LD (low density, rich in reticulocytes), D (dense), and HD (high density, rich in irreversibly sickled cells).
Microfluidic assays
The microfluidic filtering design is based on eight mechanical filtering units associated in parallel and connected together with a microchannel network. Each filtering unit is composed of five par- allel rows comprising pillars of 15 μm diameter separated by slits
of 10, 8, 7, 6 or 5 μm (Figure 1A) or four parallel rows with slits of 5, 4, 3 or 2 μm (Online Supplementary Figure S1). Side flow is ren- dered possible in the device, the U form filter zone comprises pil- lars with a 5 μm gap between them. In order to reduce the hydraulic resistance of the full design, the microchannel network is 25 μm-high compared to the 5 μm height of each filtering unit (Figure 1A). The microfluidic device was made of polydimethyl- siloxane (PDMS, Sylgard), a silicone elastomer,29 using standard microfabrication and molding. The mold was fabricated by the micro-patterning of two successive SU8 photoresist layers (Microchem, Newton, MA) to obtain a two-levels negative mold on a 4-inch Silicon substrate. The SU8 layers thicknesses were 5 μm and 25 μm, corresponding respectively to the height of the filtering units and the microchannels network. A mixture of PDMS and curing agent was poured on the SU8 mold, and reticu- lated at 75°C for 2 hours. Access through-holes were then punched, using biopsy punchers (diameter of 1.5 mm). The PDMS device, with open channels formed on one of its sides, was then assembled to a microscope coverslip, using O2 plasma activation (30 W, 300 mT, 20 s) to achieve a covalent bonding. Luer (TM) connectors were then inserted at the inlet and outlet of this microfluidic device, to achieve the sample injection with the flow controller.
For each assay, 10 μL of RBC pellet were stained with either PKH67 fluorescent Cell Linker Kit (green) or PKH26 fluorescent Cell Linker Kit (red) according to the manufacturer’s instructions (Sigma Aldrich). A 1% hematocrit solution in CellStab containing equal concentration of green and red stained RBC were loaded in the input well of the chip and perfused at constant pressure (250 mBar) using an MFCSTM-EZ-1C pump (Fluigent). RBC trapping within each filtering unit was monitored over time by sequential fluorescence images acquired using an inverted AxioObserver Z1 microscope coupled with a high resolution AxioCam MRm Rev.3 camera (Carl Zeiss). Green and red fluorescent RBC were visual- ized using the 470 and 555 nm Colibri LED (Carl Zeiss), respec- tively. Images were then analyzed using ImageJ software.30
Flow adhesion assays and red blood cell counting
RBC adhesion to Laminin 521 was evaluated under flow condi- tions using capillary flow chambers. Recombinant human Laminin 521 (BioLamina, Sundbyberg, Sweden) at 5 ng/μL was immobilized in Vena8 Endothelial+TM biochips (internal channel dimensions: length 20 mm, width 0.8 mm, height 0.12 mm). RBC were perfused at 5.107 RBC/mL for 10 min at 0.5 dyn/cm2 and 6 min washouts were performed at 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 dyn/cm2 using the ExiGoTM pump (Cellix Ltd, Dublin, Ireland). After each wash, images of adherent RBC were taken using the AxioObserver Z1 microscope (10x objective) (Carl Zeiss, Le Pecq, France). Adherent RBC were counted on each field using Image J.
The number of immobile RBC was assessed by using the Image Calculator option of the Image J software.30 The picture of one area at 2 dyn/cm2 was combined to the picture of the same area taken at 3 dyn/cm2. On the newly created image, immobile cells appeared in dark grey whereas cells present on only one of the two combined images appeared in light grey. Dark grey objects were counted with Image J software after setting an appropriate threshold.
Control blood sample oxidation
Control RBC were washed with phospate buffered saline (PBS) 1X (Thermo Fisher), suspended at 20% hematocrit in either PBS or 270 μM cumene hydroperoxide (SIGMA-ALDRICH) and incubat- ed for 2 hours with constant mild shaking. After incubation, RBC suspensions were centrifuged, the supernatant was discarded and the RBC pellets were used for subsequent experiments.
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