This study reports three important new findings. First, we present data supporting a novel anti-inflammatory strategy in vivo through using a minor groove binding drug to specifically interfere with DNA-protein binding in a targeted manner. Second, we demonstrate an important role for the architectural transcription factor HMGA1 in facilitating full induction of P-selectin promoter transactivation and inflammatory-induced gene expression. Third, we demonstrate that using transcriptional regulation to target select, similarly regulated inducible genes with common promoter motifs can be effective in improving outcomes in murine models of critical illness. Furthermore, our data supports the intriguing concept that minor groove binders can serve as an important in vivo tool to dissect molecular mechanisms of inflammatory disease processes.
Our previous work employed MGBs in vitro 
and in vivo 
to confirm an important role for TF-binding to AT-rich DNA regions in transactivation of the NOS2 promoter and in attenuating mortality and hypotension during murine endotoxemia. Numerous genes critical for the inflammatory cascade share similar promoter regulatory regions with NOS2, and we therefore hypothesized that MGBs would attenuate the inflammatory response through similarly interfering with TF-binding to AT-rich DNA regions of promoters of genes critical for the inflammatory response. Recruitment of inflammatory cells to the tissue occurs via a complicated cascade of events, each step of which is highly regulated 
. Leukocytes traversing the vasculature at rapid speed are lured to the activated endothelium through initial tethering and rolling mediated predominantly by members of the selectin family of adhesion molecules (P-selectin, E-selectin, and L-selectin). Leukocytes are then activated with subsequent firm adhesion to the endothelial surface facilitated in large part through interaction of immunoglobulin family members (on endothelial cell surface) with integrins (on leukocytes). Transmigration of leukocytes across the endothelial surface and into the tissue is likely mediated by a gradient of chemoattractants.
Given the role of MGBs in attenuating inducible gene expression 
and the observed reduction of rolling fraction and sticking efficiency of leukocytes to the endothelial surface (), we focused our studies on inducibly expressed genes previously demonstrated to play a role in the early tethering and rolling phases of leukocyte-endothelial surfaces. Therefore, we examined the effects of MGBs on P-selectin and E-selectin, both of which are inducibly expressed in endothelial cells (
, ). Of note, L-selectin is constitutively expressed on leukocytes 
. While numerous members of the adhesion molecule families provide a contributory role toward leukocyte sticking and rolling 
, studies of knockout animals have revealed that P-selectin plays the most critical and pronounced role in early leukocyte tethering and rolling 
. Therefore, the fact that induction of P-selectin can be selectively attenuated in this model () represents an important example of the potential in vivo
benefits of exquisite transcriptional control. Similar to the observed effects of MGBs on NOS2 in this model 
, inflammatory cell recruitment and P-selectin induction was decreased but not entirely abolished with MGB treatment (). Interestingly, other investigators have reported that NOS2 does not play a role in regulating expression of endothelial adhesion molecules, suggesting that our findings regarding P-selectin are independent of the regulation of NOS2 expression by Dist A in this model 
. Furthermore, P-selectin exhibits constitutive as well as inducible expression within endothelial cells 
such that the effect of leukocyte-endothelial interactions attributable to P-selectin is reduced but not eliminated with MGBs. Thus, the ability to selectively control gene transcription through interfering with TF-DNA binding presents the potential for a “titratable” anti-inflammatory effect, versus the “all or none” effect of more traditional anti-inflammatory approaches.
Interestingly, P-selectin is fairly unique as an adhesion molecule, as it is expressed not only in endothelial cells, but also in platelets, and roles of P-selectin in different locations has been a matter of recent debate. While numerous studies reported an important role for endothelial P-selectin expression in lung and liver inflammation and physiologic injury during endotoxemia 
, more recently, interest has arisen in the importance of platelet P-selectin expression in development of acid-induced lung injury 
. While we cannot fully exclude the role of platelet P-selectin in our studies, examination of histologic sections () and our studies in endothelial cells in vitro
(–) support a significant contribution of endothelial P-selectin to our observations. Moreover, a recent study reported that P-selectin glycoprotein-ligand-1 regulates lung neutrophil recruitment independently of circulating platelets in a murine model of abdominal sepsis (cecal ligation and puncture model 
). In aggregate, our findings in conjunction with those in the literature support the intriguing possibility that mechanisms of tissue neutrophil recruitment during indirect lung injury (e.g., systemic endotoxin, abdominal sepsis) are distinct from those that predominate during direct injury. Tissue neutrophil recruitment during indirect lung injury might rely more heavily on endothelial P-selectin expression, while platelet P-selectin expression may play a more prominent role during direct lung injury.
Our results indicate that MGBs can serve as a useful tool to probe the functional effects of targeted DNA sequences in complicated biological systems in vivo. Through observing the effects of MGBs in inhibiting HMGA1-binding to a targeted AT-rich region of the P-selectin promoter and in demonstrating the effects of the dominant-negative HMGA1 construct in attenuating induction of the P-selectin promoter by TNF-α, we were able to derive an important role for HMGA1 in regulating P-selectin promoter induction (–). We acknowledge that examination of the effect of MGBs on P-selectin induction in the presence of HMGA1 knockdown would provide more direct evidence that HMGA1 expression is essential for the observed effects of DistA. However, these experiments were not technically feasible, given the interference of the siRNA reagents with the MGB agents.
Other investigators have elegantly demonstrated in Drosophila that targeted minor-groove binding drugs can be fed to flies that interfere with binding of the Drosophila HMGA1 orthologue (termed D1) to specific AT-rich DNA sequences and result in specific gain- and loss-of-function phenotypes 
. In these studies, small cell-permeable molecules were synthesized based upon the existing known structure of Distamycin A and were designed with the goal of developing improved tools to elucidate the role of architectural DNA regions in biology in model systems 
. In recent years, there has been increasing interest in development of derivatives of MGBs as human chemotherapeutics to allow targeted delivery of DNA-modifying agents 
. Furthermore, improvements in techniques to elucidate molecular structure has led to a growing literature on detailed characterization of MGB-DNA binding as well as on development of novel compounds with optimized DNA-binding and functional properties 
. Our data supports the premise that novel small molecules interfering in a targeted way with sequence- and conformation-specific DNA binding can be studied at the molecular and physiologic level in higher order organisms subjected to models of human disease. Such approaches hold promise for development of novel treatment strategies for critical illness. To our knowledge, the present study and our prior work 
represent the first in vivo
applications of MGBs to murine models of critical illness.
In summary, we now demonstrate that MGBs can interfere in a targeted manner with HMGA1 binding to the P-selectin promoter in vivo
, resulting in attenuated P-selectin induction and decreased lung and liver inflammation during murine endotoxemia. These findings, in combination with our previous data showing improvement in mortality and hypotension during murine endotoxemia attributed to attenuated NOS2 induction 
, supports the interesting possibility that there exist select genes regulated by common promoter motifs that can be advantageously regulated to improve outcomes from critical illness. With a growing appreciation of conserved regulatory motifs throughout the human genome and with increasing ability to catalogue this data 
, there exists a real possibility of molecularly targeted treatment strategies that can be applied in individualized ways to complex human disease. We acknowledge that MGBs may have other effects on an organism that remain to be characterized. However, our work represents implementation of MGBs as a molecular tool to derive in vivo
biological characterization of critical illness that ultimately can be applied to the development of novel therapeutics in a field where effective treatment approaches are desperately needed.