Identifying regulators of endothelial immunesurveillance by patrolling monocytes to promote vascular health

Línea de investigación

Matrix metalloproteinases in Angiogenesis and Inflammationn.

Descripción

Patrolling monocytes (PMo) are endowed with the ability to crawl along the apical side of the endothelium in search of damage to promote its repair. Our group proposed that PMo crawling & search mode are coupled shifting from diffusive random in healthy conditions to Levy-like walk in the presence of damaged endothelium (Moreno-Cañadas et al., Front Immunol 2021). Maintaining the endothelium in good condition is essential to prevent cardiovascular and metabolic disorders and promote healthy aging. However, the damaging cues exposed by the endothelium to switch PMo crawling & search mode and the mechanisms underlying restoration of endothelial homeostasis remain largely unknown. In this line, our team recently found that the absence of the MT4-MMP protease increased intravascular PMo surveillance (Clemente et al., Nat Commun 2018).

We hypothesize that deciphering endothelial damage signals and their crosstalk with PMo will help identify regulators whose targeting may boost the protective actions of PMo in the vasculature.

The TFM student will be trained in a variety of techniques to develop the following objectives and work plan:

1. Search for endothelial cell stimulators that promote PMo Lèvy-like walk. Mouse lung endothelial cells (MLEC) will be stimulated with factors related to inflammation, hypoxia or mechanical forces and then mouse PMo-like cells will be added to Ibidi plates. Co-cultures will be recorded by multidimensional microscopy and single-cell image analysis and hierarchical clustering will be performed with ImageJ and R to characterize cell motility patterns according to Lèvy-like walk descriptors.
2. Characterizing the mechanisms by which PMo recognize endothelial damage. Candidate-based damage signals will be explored in MLEC stimulated with different agents by staining fixed and live cells with Annexin probes and visualizing them with super-resolution & time-lapse confocal microscopy and 3D reconstruction (Imaris®). The impact of their blockade on PMo Lèvy-like walk will be analyzed by multidimensional microscopy as in 1. Activated MLEC will be labeled with lipophilic probes, co-cultured with PMo for 3 hours and possible promotion of endothelial cell repair by PMo engulfment analyzed by conventional & image flow cytometry and quantified with FlowJo & IDEAS software.

This project will lay the foundation for future host-directed PMo-based immunotherapies to prevent endothelial dysfunction and reduce cardio-metabolic diseases.

Contacto

Alicia García Arroyo.

Correo electrónico: agarroyo@cib.csic.es.

Centro de Investigaciones Biológicas Margarita Salas (CIB Margarita Salas).

Número de plazas ofertadas: 1.