Conditioned Media from Keratinocyte Growth Factor Modified Mesenchymal Stromal Cells Reduces Pulmonary Vascular Permeability
Authors: Pati Shibani , Croze Roxanne , Gibb Stuart , Potter Daniel , Fang Xiaohui , Matthay Michael A 
Published: November 2023

Background

There is an unmet need for new therapeutics to treat Acute Respiratory Distress Syndrome (ARDS), an important cause of morbidity and mortality in critically ill patients. Bone marrow‐derived mesenchymal stromal cells (MSC) attenuate acute lung injury (ALI) in preclinical models of ARDS through secretion of soluble factors into the conditioned media (CM) that act in a paracrine fashion. Keratinocyte growth factor (KGF) is secreted by MSC and mediates several of the beneficial effects in ALI preclinical models. Thus, the objective for this study was to transfect MSC with the KGF gene to enhance the beneficial effects of conditioned media (CM) derived from transfected MSC on endothelial cell permeability and pulmonary vascular injury in hemorrhagic shock. The long term goal of these efforts would be to utilize gene enhanced MSC CM in lieu of MSC.

Hypothesis

The beneficial effects of MSC CM on pulmonary and systemic vascular permeability can be enhanced through gene modification of MSC with transfection of the KGF gene.

Materials and Methods

Gene modification of MSC: To modify bone marrow‐derived MSC with a constitutive KGF expressing plasmid, nucleofection was conducted using the Amaxa Nucleofection System (Lonza, MD) and varying doses of KGF plasmid (0.5–3.0 ug). Endothelial Permeability: The integrity of EC (HUVEC) monolayers and trans‐endothelial resistance (TEER) was measured using an electric cell‐substrate impedance sensing system (ECIS) (Applied BioPhysics, Troy, NY). Hemorrhagic Shock (HS) and Trauma model of ARDS: Male C57BL6 mice, 8–12 weeks old were subjected to a 2.5‐cm midline laparotomy. Using an established model of HS, mice were bled to a mean arterial blood pressure of 35 mm Hg for 90 minutes. Mice were resuscitated MSC and MSC CM in 200 ul of saline. Pulmonary vascular permeability was measured using an Alexa Flour dextran 10 kDa injected into the pulmonary artery one hour prior to sacrifice.

Results

Nucleofection of MSC with KGF plasmid increased protein concentrations of KGF in the CM by 4‐fold. In vitro, KGF modified MSC CM significantly enhanced endothelial monolayer resistance and reduced permeability compared to control MSC CM as measured by ECIS. Area under the curve measurements: CM from control media (0.63 ± 0.011), CM from GFP transfected MSC (0.66 ± 0.0029), CM from mock transfected cells (0.65 ± 0.013) and CM from KGF transfected cells (0.68 ± 0.0076), p<0.05 GFP versus KGF (Figure). In vivo, both MSC and KGF enhanced MSC CM demonstrated significant potent effects in attenuating pulmonary vascular permeability induced by HS and trauma with a 35% reduction in accumulation of the 10kDa dextran in the extravascular spaces of mouse lungs compared to controls (P<0.05).

Conclusion

KGF‐enriched conditioned media from MSC reduces in vitro endothelial permeability and also in vivo lung permeability from hemorrhagic shock. KGF‐enriched conditioned media from MSC may provide an alternative to treat increased lung vascular permeability from acute lung injury. Use of a cell‐free therapeutic may have logistical, safety and regulatory advantages for implementation in patients with ARDS.

Support or Funding Information

NHLBI UC‐CAI 20130924SFM