Therapeutic Potential of Beta-Nicotinamide Adenine Dinucleotide to Reverse Endothelial Barrier Dysfunction in Sickle Cell Mouse Model

Background: The pulmonary endothelium is a metabolically active monolayer on the luminal surface of the lung vasculature. It regulates a variety of functions including the passage of macromolecules and fluid between the blood and lung interstitial tissue.  Inflammatory agents such as bacterial endotoxin lipopolysaccharide (LPS) cause lung endothelial cells (EC) dysfunction leading to pulmonary edema predisposing to acute chest syndrome, a leading cause of death in sickle cell disease (SCD); therefore, a drug which repairs the damaged EC barrier function might be of therapeutic potential. Recently we demonstrated that β-nicotinamide adenine dinucleotide (β-NAD) reverses LPS-induced EC barrier dysfunction in a model of acute lung injury (Exp Lung Res2012, 38, 223-32). In this study, we investigated the protective effect of β-NAD against the damaging effects of LPS using primary ECs from human and sickle cell mouse lungs as well as survival studies using the preclinical sickle cell mouse model.

Methods: Early passaged human lung microvascular EC (HLMVEC) were obtained from Lonza and mouse lung microvascular EC (MLMVEC) were isolated from the Townes knock-in homozygous sickle cell (SS) and heterozygous sickle trait (AS) transgenic mice. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) technique. Western blot was done to measure phosphorylation of proteins involved in the regulation of EC barrier function. Survival studies were performed in SS mice treated with intraperitoneal (IP) LPS (0.05 mg/kg) alone or combined with β-NAD (11 mg/kg); normal saline served as a control. Lungs were harvested at 10-12 hrs from the LPS group and at 24 hrs from saline or LPS/β-NAD groups and stained with Hematoxylin and Eosin for to assess interstitial edema and myeloperoxidase as a marker of neutrophil infiltration.

Results: Barrier function measurements using ECIS of HLMVEC indicate that β-NAD (100 µM) enhanced basal EC barrier function by 50-60%. In addition, β-NAD attenuated LPS-induced EC gap formation and actin stress fibers. In human lung endothelium, myosin light chain phosphatase (MLCP) plays a crucial role in the maintenance of the EC barrier integrity. Furthermore, LPS mediates MLCP inactivation by phosphorylating the MLCP regulatory subunit MYPT1 and cytoskeletal targets ERM (Ezrin, Radixin, and Moesin) and CaD (Caldesmon). Western blot analysis demonstrated that β-NAD reversed LPS-induced phosphorylation of MYPT1, ERM and CaD proteins.  To determine a functional role, we performed studies using 10 nM MYPT1 siRNA treatment followed by β-NAD (100 μM) for 5 hrs. We observed a 50% attenuation of β-NAD enhanced barrier function suggesting MYPT1 is involved in the mechanism of action for β-NAD. Subsequent studies with SS-MLMVEC demonstrated β-NAD mediated a 40% improvement in barrier function which was comparable to HLMVEC. Further, LPS treatment for 16 hrs produced a 25% decrease in barrier function in SS-MLMVEC compared to a 7% decrease in AS-MLMVEC suggesting impaired EC barrier function in SS lungs. Finally we performed survival studies to determine the ability of β-NAD to protect against LPS-induced mortality in SS mice. In dose optimization studies of LPS (0.1-1.0 mg/kg/dose), SS mice survived for 10-12 hrs with the 0.05 mg/kg LPS dose.  By contrary, all LPS doses tested had no effect on AS mice survival. Based on this result, SS mice were given IP injections of saline (3 mice), LPS (4 mice) or LPS/β-NAD (4 mice) and survival rates were monitored. The saline or LPS/β-NAD treated mice survived for 24 hrs (100%) compared with LPS mice which died by 10-12 hrs (p=0.01) supporting a protective effect of β-NAD. Histological assessment of the lung tissue showed increased interstitial edema by Hematoxylin and Eosin stain and neutrophil infiltration by myeloperoxidase in the LPS-treated lungs; these changes were much less prominent in the lungs from LPS/β-NAD treated SS mice.

Conclusions: Our data support a role of MLCP and its cytoskeletal targets in the protective effects of β-NAD against LPS-induced HLMVEC barrier dysfunction. Studies using SS-MLMVEC suggest that SS mice have impaired barrier function and greater susceptibility to LPS toxicity. However, β-NAD treatment improved survival of SS mice supporting the therapeutic potential of this agent to treat the complication of acute chest syndrome in SCD.