Application of physiomimetic stimuli for realistically reproducing pathological hypoxic and micromechanical cell environments

Abstract

HYPOTHESIS 1:

The frequency and the plateau values displayed by OSA-related IH patterns are important to understand the endothelial dysfunction triggered by OSA. AIM 1:

A) General:

To evaluate the contribution of frequency and magnitude of IH in an in vitro model of aortic wound closure. B) Specific:

1) To build up an in vitro device that enable the modulation of frequency and magnitude of IH.

2) To quantify the wound closure index of a human aortic endothelial monolayer cultured under relevant constant hypoxic and normoxic values.

3) To compare and contrast the effect on the wound closure index of different cycling rates and plateau values of IH cycles and the contribution of the maximum and minimum values.

HYPOTHESIS 2:

The oxygen gradients observed in the tumor microenvironment could affect the cross-talk between macrophages and tumor cells leading to macrophage recruitment and cancer malignancy by creating an immune-permissive tumor microenvironment. AIM 2:

a) General:

To study the potential role of the O2 gradient, found in solid tumors, on macrophage recruitment and phenotype and the importance in cancer development. b) Specific:

1) To design and construct a co-culture in vitro model that enable simultaneously culture of two types of cells under a different oxygenation paradigm.

2) To quantify macrophage recruitment and evaluate the polarization of macrophages to M1/M2 phenotypes when co-cultured with tumor cells exposed to differential levels of oxygen.

3) To evaluate the cell growth of cancer cells and quantify the expression of relevant genes related to the macrophages roles in the tumor microenvironment.

HYPOTHESIS 3:

The biophysical preconditioning of LMSC with physiological lung ECM cues could improve their therapeutic potential for the treatment of ARDS. AIM 3:

A) General:

To study the effects of biophysically fostered LMSC in a rat ventilator-induced lung injury (VILI) model of ARDS. B) Specific:

1) To build up an in vitro model to culture LMSC on a lung decellularised scaffold and subjected to cyclic stretch.

2) To evaluate the effect of Preconditioned LMSC on respiratory mechanics and pulmonary edema of rats subjected to VILI.

3) To assess the effect of Preconditioned LMSC on lung injury markers and immune cells from the BAL.

4) To study the capacity of Preconditioned LMSC to engraft to the ARDS rat lung after transplantation.

Hypothesis 4

Culturing LMSC in 3D within lung ECM-derived hydrogels could provide a more similar biophysical milieu to the native tissue which could improve their therapeutic potential affecting relevant mechanobiology outcomes. Aim 4

General

To develop a 3D hydrogel model based on lung ECM cues to study therapeutic relevant outcomes on LMSC with the ultimate purpose of developing improved therapeutic approaches for lung inflammatory diseases. Specific

1) To develop and characterize a material derived from the lung pig ECM to culture LMSC in 3D.

2) To evaluate macroscopically the interaction between the hydrogel and the LMSC.

3) To study cell adhesion and quantify CXCR4 gene expression of LMSC cultured in this 3D model.

4) To evaluate inflammatory markers of LMSC, previously cultured in lung hydrogels, in a co- culture epithelial model of LPS-induced acute lung injury.