Stabilization of Hypoxia-Inducible Factor-1 Alpha Augments the Therapeutic Capacity of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Pneumonia

Bone marrow-derived mesenchymal stem cells (MSCs) have therapeutic effects in experimental models of lung injury. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcriptional regulator that influences cellular metabolism, energetics, and survival under hypoxic conditions. The current study investigated the effects of stabilizing HIF-1α on the therapeutic capacity of MSCs in an experimental mouse model of bacterial pneumonia. HIF-1α stabilization was achieved by the small molecule prolyl-hydroxlase inhibitor, AKB-4924 (Aerpio Therapeutics, Inc.), which blocks the pathway for HIF-1α degradation in the proteosome. In vitro, pre-treatment with AKB-4924 increased HIF-1α levels in MSCs, reduced the kinetics of their cell death when exposed to cytotoxic stimuli, and increased their antibacterial capacity. In vivo, AKB-4924 enhanced MSC therapeutic capacity in experimental pneumonia as quantified by a sustainable survival benefit, greater bacterial clearance from the lung, decreased lung injury, and reduced inflammatory indices. These results suggest that HIF-1α stabilization in MSCs, achieved ex vivo, may represent a promising approach to augment the therapeutic benefit of these cells in severe pneumonia complicated by acute lung injury.

and sepsis as there are no proven pharmacological therapies in this field (4)(5)(6)(7)(8)(9). MSCs have a number of biological properties that lend them to producing a favorable outcome in lung injury and sepsis including immunomodulation, secretion of epithelial and endothelial growth factors, and augmentation of host defense to infection (6,10,11). However, the clinical benefits of MSCs in trials have been modest, which may be due to a lack of sustained benefit given MSC death and clearance under inflammatory conditions in vivo. It has previously been shown that non-viable MSCs exert no therapeutic benefit (5). Thus, methods to enhance MSC survival and augment their therapeutic capacity should improve their efficacy in clinical lung injury and sepsis. Hypoxia-inducible factor-1 alpha (HIF-1α) is an important transcriptional regulator that controls many cellular processes under hypoxic conditions, and the injured lung represents a low-oxygen tension environment that presents a metabolic stress to cells introduced into that space. Prior efforts suggested that stabilization of cellular HIF-1α levels could increase the therapeutic function of MSCs in cardiac and vascular injury models (12)(13)(14). Consequently, we hypothesized that HIF-1α stabilization in MSCs would enhance their therapeutic efficacy in experimental lung injury and pneumonia, potentially by improving cell survival in the face of inflammatory, cytotoxic stimuli. To that end, we pharmacologically stabilized HIF-1α in MSCs using AKB-4924 (Aerpio Therapeutics, Blue Ash, OH, USA) given our previous experience with the selective potency of this compound (15)(16)(17).

MethoDs isolation, characterization, and culturing of Mscs
Mouse MSCs were isolated from 8-to 10-week old male C57BL/6J mice and characterized as published before (8). MSCs were then cultured using MEM-alpha media (Gibco, catalog #12561) with 15% FBS (Gibco, catalog #12662-029) and 1% Pen/Strep/ l-Glutamine and used for in vitro and in vivo experiments from passages 5 to 10.

hif-1α stabilization in Mscs and Western Blotting
Mesenchymal stem cells were incubated in the presence of AKB-4924 in a 12-well plates for 4 and 24 h to determine the optimal time and concentration for HIF-1α stabilization in MSCs. AKB-4924 was used at 10 and 100 µM in MEM-alpha supplemented with 5% FBS. MSCs were then lysed and the protein fraction isolated, quantified, and analyzed for HIF-1α expression by Western blotting (see Supplementary Material for details). Based on the data, AKB-4924 was used at 100 µM for 4 h on MSCs to stabilize HIF-1α in most in vitro and in vivo studies.

In Vitro Bacterial Killing studies
To determine if AKB-4924 enhances MSC killing of bacteria, separate assays were done with live MSCs and MSC-derived conditioned media in the presence of Escherichia coli (see Supplementary Material). Mouse cathelicidin-related antimicrobial protein (CRAMP ELISA, MyBioSource, catalog #MBS280706) was specifically measured to determine if it accounted for the antimicrobial effects induced by AKB-4924. Gene expression for CRAMP was quantified using qPCR as outlined below.
In Vitro cell Death and caspase 3/7 activity To measure the effect of AKB-4924 on MSC death when exposed to cytotoxic, inflammatory stimuli, studies were done to measure caspase 3/7 activity in a plate-based assay (Promega, catalog #G7790). TNF-α and cycloheximide were chosen as the stimuli since this combination resulted in the most reproducible quantity of cell death for MSCs, and it has been published as an in vitro method to model cell death in an inflammatory environment (18,19) (see Supplementary Material).

rNa isolation and qpcr
In vitro studies were done to determine if AKB-4924 regulated expression of selected genes (CRAMP, Oct4, TWIST) in MSCs that could account for the observed in vitro and in vivo effects. RNA was isolated and qPCR was carried out using standard procedures (see Supplementary Material).

In Vivo E. coli pneumonia Model and experimental Design
All mice used for these experiments were male C57BL/6J (Jackson Labs) between the ages of 10 and 15 weeks of age. All experiments were approved by the University of California, San Diego (UCSD) Institutional Animal Care and Use Committee, and mice were housed in a UCSD facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. The general experimental design that we followed is as previously published (5,8)

assessment of Lung injury, inflammation, and Bacterial Burden
Lung injury was assessed by histological methods and scored using a previously published method (20). Markers of inflammation and permeability were measured in the bronchoalveolar lavage (BAL) fluid (5,8), and bacterial burden was calculated from whole lung homogenate (see Supplementary Material).

statistical analysis
The majority of the data is presented as mean ± SD for each group analyzed. An unpaired, two-sided Student's t-test was used for comparisons between sets of data. For sets of data with a small sample size (total n < 20), a Mann-Whitney U test was used. If multiple groups of data were compared simultaneously, an ANOVA was used. Survival data were analyzed using a log-rank test. A p-value <0.05 was used for statistical significance for all analyses.  Figure 1A). MSCs treated with AKB-4924 exhibited significantly reduced cell death, as measured by caspase 3/7 activity, when exposed to TNF-α and cycloheximide ( Figure 1B).

aKB-4924 enhances the antibacterial capacity of Mscs
In vitro, AKB-4924 was able to significantly improve MSC-based reduction of viable E. coli. The effect of AKB-4924 occurred under both basal and TNF-α stimulated conditions ( Figure 1C). Conditioned media from AKB-4924 stimulated and TNF-α + LPS stimulated MSCs demonstrated an approximate 20% reduction in viable E. coli compared with conditioned media from unstimulated MSCs (Figure 1D). This suggests that release of an antimicrobial factor into the conditioned media may account for part of the increased bacterial killing by MSCs that is induced by AKB-4924. We hypothesized that this factor may be mouse CRAMP given previous literature demonstrating that the human cathelicidin antimicrobial protein LL-37 is a potential transcriptional target of HIF-1α, and that human MSCs exert antibacterial effects via LL-37 secretion (10,21). However, under the conditions utilized in this study, we did not detect a significant increase in CRAMP protein secretion in HIF-1α stabilized MSCs (Figure 1E).

aKB-4924 improves Msc-Derived therapeutic capacity In Vivo
To determine if the in vitro benefits with AKB-4924 described above translated into greater MSC-derived therapeutic capacity in vivo, the experimental design using an E. coli pneumonia model outlined in Figure 2A was utilized. While both unstimulated and AKB-4924 stimulated MSCs exerted significant survival benefits at 72 h (Figure 2B), only MSCs incubated with AKB-4924 conferred sustained protection against mortality over the course of 7 days (Figure 2C). Bacterial clearance from the lung at 24 h post-infection was significantly impro ved with MSCs incubated with AKB-4924 as well ( Figure 2D). In addition, HIF-1α stabilized MSCs led to a significant reduc tion in inflammatory indices such as BAL myeloperoxidase (MPO) and macrophage inflammatory protein-2 (MIP-2) levels 24 h after infection (Figures 2F,G, respectively), though there was not a significant reduction in the total BAL cell count ( Figure 2E) or BAL albumin concentration ( Figure 2H). BAL CRAMP was measured to see if it correlated with the reduction in bacterial burden seen in Figure 2D, but the increase in CRAMP observed with HIF-1α stabilized MSCs did not reach statistical significance (Figure 2I). The improvements in bacterial clearance and inflammation were associated with a reduction in lung injury at 48 h postinfection, as assessed by histological methods, that was more pronounced in mice treated with HIF-1α stabilized MSCs (Figures 2J,K).

DiscUssioN
Mesenchymal stem cells have been extensively studied as a potential therapy for severe lung injury and sepsis and have shown promise in several pre-clinical models (4)(5)(6)(7)(8)(9)(10)(11). However, strategies to improve the survival of MSCs in inflammatory environments and thus augment their therapeutic potential are needed. This proof-of-principle study sought to enhance the therapeutic potential of MSCs in experimental lung injury due to pneumonia by stabilizing the transcription factor HIF-1α with the pharmacological agent AKB-4924. Results from this study substantiated our hypothesis by demonstrating that AKB-4924 improved: (a) MSC survival under in vitro cytotoxic conditions; (b) MSC antibacterial activity in vitro; and (c) MSC-derived therapeutic capacity in vivo with reduced mortality, bacterial burden, inflammation, and lung injury. Though, it is interesting to note that while BAL MPO levels were reduced, total BAL cell counts were not in this study. This discordance may due to a greater effect on neutrophil degranulation as opposed to absolute neutrophil recruitment to the alveolar space. Also, the lack of reduction in BAL albumin at 24 h is not concordant with the other parameters measured, which may be because it represents a summation of permeability over the entire time period and is not sensitive enough to detect changes that develop later in the timeframe being studied. Nevertheless, the overall findings suggest that methods to stabilize HIF-1α in MSCs could be implemented in order to boost the therapeutic effect achieved in critically ill patients with lung injury, and are consistent with recent promising results in cardiac and vascular disease models (12)(13)(14).
Mesenchymal stem cells have been tested in several hundred clinical trials to date targeting a wide range of clinical diseases, but their clinical efficacy has not been reproducibly robust to date (22,23). One of the potential explanations that has been suggested is the relatively short half-life of MSCs in vivo (24,25). HIF-1α represents an intuitive target to augment survival of MSCs in lung injury applications since the injured lung is a hypoxic environment requiring metabolic adaptations. Recent studies in experimental models of ischemia-reperfusion and radiation-induced lung injury have shown that hypoxic preconditioning of MSCs enhances their therapeutic efficacy (26,27). The mechanisms demonstrated include improved MSC survival and antioxidant ability.
In this study, HIF-1α stabilization in MSCs with the use of AKB-4924 resulted in significantly improved MSC survival under cytotoxic conditions and MSC-derived therapeutic capa city in vivo. While improving MSC survival is likely an important contributor to the augmented biological effect achieved with HIF-1α stabilized MSCs, there are other possible mechanisms to consider. We provide some preliminary data that HIF-1α stabilization augments the antibacterial property of MSCs, and it is possible that HIF-1α stabilization in MSCs may be boosting other biological effects of MSCs such as growth factor secretion and immunomodulation. We also tested the possibility that HIF-1α stabilization could keep MSCs in an undifferentiated, "stem-like" state that permits them to retain their reparative properties for a longer duration (28). However, screening qPCR analyses to determine if HIF-1α stabilization upregulated-specific genes involved in maintaining an undifferentiated MSC phenotype (Oct4, TWIST) were unable to detect a significant difference compared with unstimulated MSCs ( Figure S1 in Supplementary Material). Finally, HIF-1α stabilized MSCs may be modulating the survival and function of other cell types that are known to be present in the injured lung such as alveolar epithelial cells, endothelial cells, neutrophils, and macrophages. These other potential mechanisms remain the focus of ongoing and future investigations.
While we used a small molecule, AKB-4924, to stabilize HIF-1α in MSCs there are other potential methods that could be used to achieve this goal. Previous studies have used hypoxic preconditioning (i.e., growing MSCs under hypoxic conditions) to augment HIF-1α expression. In addition, genetic editing could be applied to MSCs in order to inactivate the prolyl hydroxylase enzymes responsible for HIF-1α degradation under normoxic conditions. However, genetic editing may carry an increased risk of malignant transformation of MSCs due to sustained dysregulation of HIF-1α expression, particularly since HIF-1α has been implicated in tumor development and invasiveness (29)(30)(31). In this regard, the use of AKB-4924 affords the advantage of stabilizing HIF-1α for a defined time period that is determined by its own half-life. For acute inflammatory processes, such as lung injury due to bacterial pneumonia, even transient stabilization of HIF-1α can lead to significant beneficial outcomes as we observed.
In summary, stabilization of HIF-1α in MSCs, with the use of AKB-4924, significantly boosts MSC-derived therapeutic capacity in an E. coli model of bacterial pneumonia. Mechanistically, this may be due, in part, to improved MSC survival under cyto toxic conditions. This study and other recent publications suggest that strategies to stabilize HIF-1α should be incorporated into MSC-based clinical trials for critically ill patients with lung injury.

ethics stateMeNt
All experiments were approved by the University of California, San Diego (UCSD) Institutional Animal Care and Use Committee (IACUC), and mice were housed in a UCSD facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).