Abstract
BackgroundAcute lung injury (ALI) and in its severe form, acute respiratory distress syndrome (ARDS), results in increased pulmonary vascular inflammation and permeability and is a major cause of mortality in many critically ill patients. Although cell-based therapies have shown promise in experimental ALI, strategies are needed to enhance the potency of mesenchymal stem cells (MSCs) to develop more effective treatments. Genetic modification of MSCs has been demonstrated to significantly improve the therapeutic benefits of these cells; however, the optimal vector for gene transfer is not clear. Given the acute nature of ARDS, transient transfection is desirable to avoid off-target effects of long-term transgene expression, as well as the potential adverse consequences of genomic integration.MethodsHere, we explored whether a minicircle DNA (MC) vector containing human angiopoietin 1 (MC-ANGPT1) can provide a more effective platform for gene-enhanced MSC therapy of ALI/ARDS.ResultsAt 24 h after transfection, nuclear-targeted electroporation using an MC-ANGPT1 vector resulted in a 3.7-fold greater increase in human ANGPT1 protein in MSC conditioned media compared to the use of a plasmid ANGPT1 (pANGPT1) vector (2048 ± 567 pg/mL vs. 552.1 ± 33.5 pg/mL). In the lipopolysaccharide (LPS)-induced ALI model, administration of pANGPT1 transfected MSCs significantly reduced bronchoalveolar lavage (BAL) neutrophil counts by 57%, while MC-ANGPT1 transfected MSCs reduced it by 71% (p < 0.001) by Holm-Sidak’s multiple comparison test. Moreover, compared to pANGPT1, the MC-ANGPT1 transfected MSCs significantly reduced pulmonary inflammation, as observed in decreased levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). pANGPT1-transfected MSCs significantly reduced BAL albumin levels by 71%, while MC-ANGPT1-transfected MSCs reduced it by 85%.ConclusionsOverall, using a minicircle vector, we demonstrated an efficient and sustained expression of the ANGPT1 transgene in MSCs and enhanced the therapeutic effect on the ALI model compared to plasmid. These results support the potential benefits of MC-ANGPT1 gene enhancement of MSC therapy to treat ARDS.
Highlights
Acute respiratory distress syndrome (ARDS) was first described five decades ago in patients with tachypnea and refractory hypoxemia, with main symptoms consisted of diffuse opacities on chest radiographs [1]
We have previously reported improved therapeutic efficacy of Mesenchymal stem cell (MSC) that can be enhanced by plasmid DNA angiopoietin-1 transfection in LPS-induced Acute lung injury (ALI) in mice [9]
Minicircle DNA-transfected MSCs released significantly more ANGPT1 proteins compared to plasmid transfection Significantly higher levels of ANGPT1 proteins were detected in conditioned media of MSCs transfected with ANGPT1 coding sequence using minicircle (MC-AGNPT1) vs. plasmid vectors at 24 h post transfection (2048 ± 567 pg/mL vs. 552.1 ± 33.5 pg/mL, respectively; Fig. 1b)
Summary
Acute respiratory distress syndrome (ARDS) was first described five decades ago in patients with tachypnea and refractory hypoxemia, with main symptoms consisted of diffuse opacities on chest radiographs [1]. Mesenchymal stem cell (MSC) therapy is considered a promising intervention for treating ARDS due to the immunomodulatory and anti-inflammatory ability of these cells [7, 8]. Previous studies have demonstrated that bone marrow-derived mesenchymal stem cells (BMMSCs) can reduce pulmonary inflammation and lung permeability [9,10,11]. Leng et al have reported improved pulmonary function and symptoms of COVID-19 patients who received MSC therapy [18]. Acute lung injury (ALI) and in its severe form, acute respiratory distress syndrome (ARDS), results in increased pulmonary vascular inflammation and permeability and is a major cause of mortality in many critically ill patients. Cell-based therapies have shown promise in experimental ALI, strategies are needed to enhance the potency of mesenchymal stem cells (MSCs) to develop more effective treatments. Given the acute nature of ARDS, transient transfection is desirable to avoid off-target effects of long-term transgene expression, as well as the potential adverse consequences of genomic integration
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