Downregulation of Cytoskeletal Protein Kazrin in Lung Endothelial Cells of Preclinical Sickle Cell Disease Mouse Model - Essential Role of Endothelial Barrier Function
Authors: Siddaramappa Umapathy N , Patel Niren , Pace Betty S. , Kutlar Abdullah 
Published: November 2019

Background: Lung capillary barrier dysfunction is common to all causes of acute chest syndrome (ACS), a leading cause of morbidity and mortality in individuals with sickle cell disease (SCD). Our published data (Haematologica 2017, 102, e26-e29) demonstrated, for the first time, an impaired barrier function in freshly isolated endothelial cells (EC) from the lungs of Townes transgenic sickle cell anemia (SS) mice, compared to heterozygote (AS) or control (AA) mice. In addition, we showed a sub-stimulatory dose (0.05 mg/kg) of bacterial toxin lipopolysaccharide, which did not cause prominent acute lung injury in control AS/AA mice, was fatal to SS mice suggesting an enhanced hypersensitivity of sickle EC to infection. Hemolysis is a fundamental feature of SCD that contributes to its pathophysiology. Hemolysis results in the leakage of vast amounts of hemoglobin, and subsequently, heme into the plasma. Although, previous studies have demonstrated the detrimental effect of heme in SCD, the mechanisms of heme-mediated lung EC barrier dysfunction have not been studied. Recently, Kazrin was identified as a widely expressed protein influencing intercellular adhesion and co-localizes with the cortical actin ring to stabilize cell-cell junctions. In this study, we investigated the mechanisms of heme-induced EC barrier dysfunction related to Kazrin function, using primary EC isolated from the lungs of SCD mice and controls.
Methods: Primary EC from the lungs of SS, AS, and AA mice (6-8 weeks old) were isolated according to methods described in our recent publication. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) and Evans blue dye transendothelial permeability was done using a transwell filter assay system. Western blot was performed to measure total and phosphorylated expression levels of protein involved in the regulation of EC barrier function. Immunocytochemistry was performed using specific antibodies and actin cytoskeleton was identified using a known F-actin binding fluorescent marker Phalloidin.
Results: Our data demonstrated a significantly reduced basal expression of the Kazrin mRNA and protein in freshly isolated SS-EC compared to AS-EC or AA-EC. To determine the effects of Kazrin downregulation on the EC barrier function, we depleted the Kazrin protein in AS-EC using Kazrin specific siRNA and scrambled control siRNA. Kazrin depleted cells demonstrate an attenuated EC barrier function as evidenced by reduced trans-endothelial electrical resistance by ECIS and increased Evans blue dye leak by transwell filter assay (p<0.05). By contrast, control siRNA treated AS-EC displayed normal barrier function. Therefore, our data, for the first time, suggest that Kazrin is essential for the EC barrier function. Given the crucial role of hemolysis in SCD, we detected that clinically relevant dose of heme (5 µM; 60 min) significantly decreases the AS-EC barrier function (p<0.05) and the heme-mediated decrease of barrier function is dose-dependent. Moreover prolonged treatment of AS-EC with heme (5 µM; 24-48 hrs) downregulated the Kazrin protein levels. These results are intriguing since the SS-EC inherently show downregulated Kazrin when compared with AS-EC. Therefore, it is possible that impaired barrier function in SS-EC that we recently published could be an effect of Kazrin downregulation since hemolysis is a constant process in SCD. We also found that incubation of AS-EC with heme upregulated the efficacy of barrier disruption agent RhoA. Previous studies have indicated that activated RhoA promotes the actin stress fibers by stimulating actin-myosin contractility and increased endothelial permeability. Our immunocytochemistry data reveal that heme incubated AS-EC develop severe actin stress fibers. Studies in progress to determine the mechanistic link between Kazrin downregulation, RhoA activation and impaired EC barrier function in SS-EC.
Conclusions and Future Direction: Our findings support the notion that Kazrin participates in the EC barrier function and its downregulation cause an impaired EC barrier function. Our future studies will focus on to identify protective mechanisms of lung EC barrier function in SCD.

Disclosures
Kutlar: Bluebird Bio: Other: DSMB Member; Novo Nordisk: Research Funding; Novartis: Consultancy; Micelle Biopharma: Other: DSMB Chair; Global Blood Therapeutics, Inc. (GBT): Research Funding.