HDAC6 inhibition prevents TNF-α-induced caspase 3 activation in lung endothelial cell and maintains cell-cell junctions

Pro-inflammatory mediators such as TNF-α induce caspase activation in endothelial cells, which leads to degradation of cellular proteins, induction of apoptotic signaling, and endothelial cell dysfunction. New therapeutic agents that can inhibit caspase activation may provide protection against inflammatory injury to endothelial cells. In the present study, we examined the effects of selective histone deacetylase 6 (HDAC6) inhibition on TNF-α induced caspase 3 activation and cell-cell junction dysfunction in lung endothelial cells. We also assessed the protective effects of HDAC6 inhibition against lung inflammatory injury in a mouse model of endotoxemia. We demonstrated that selective HDAC6 inhibition or knockdown of HDAC6 expression was able to prevent caspase 3 activation in lung endothelial cells and maintain lung endothelial cell-cell junctions. Mice pre-treated with HDAC6 inhibitors exhibited decreased endotoxin-induced caspase 3 activation and reduced lung vascular injury as indicated by the retention of cell-cell junction protein VE-Cadherin level and alleviated lung edema. Collectively, our data suggest that HDAC6 inhibition is a potent therapeutic strategy against inflammatory injury to endothelial cells.


INTRODUCTION
Inflammation-mediated endothelial cell damage including inflammatory lung vascular injury is often associated with caspase activation and endothelial cell barrier dysfunction [1]. Over-production of proinflammatory mediators such as tumor necrosis factor (TNF)-α is a major cause of endothelial cell injury during inflammation [1]. Tumor necrosis factor (TNF)-α-induced caspase activation and endothelial cell dysfunction contribute to inflammatory vascular injury in endotoxemia and sepsis [1]. New agents that can prevent inflammatory injury to endothelial cells could provide therapeutic benefits.
In the present study, we investigated the effects of HDAC6 knockdown and HDAC6 inhibitors Tubastatin A and CAY10603 on TNF-α-induced caspase 3 activation in endothelial cells in vitro. We examined the effects of Tubastatin A and CAY10603 on cell-cell junction integrity and endothelial permeability in primary human lung endothelial cells. Furthermore, the effects of Tubastatin A and CAY10603 on caspase 3 activation and lung edema formation in vivo was assessed in a mouse model of endotoxemia.

HDAC6 inhibition alleviates TNF-α-induced disruption of tight junctions in endothelial cells
Caspase-3 activation is associated with endothelial cell-cell junction disruption [2][3][4]. We next conduct experiments to assess whether HDAC6 inhibition could prevent TNF-α-induced damage to endothelial cellcell junctions. We observed endothelial cells ZO-1 disassembly at tight junctions after TNF-α challenged by immunofluorescence assay. Pre-treatment with Tubstatin A prevented the disruption in HPAECs and HLMVECs ( Figure 3).

HDAC6 inhibition blocks endotoxin-induced caspase-3 activation and VE-Cadherin downregulation in the lung
To assess the therapeutic potential of HDAC6 inhibition against endothelial cell injury during acute inflammation, we examined the effects of CAY10603 on caspase-3 activation in a mouse model of endotoxemia. In our studies, endotoxin-induced caspase-3 activation in the lung tissues was significantly inhibited by CAY10603 and Tubastatin A pre-treatment. (Figure 4). Furthermore, endotoxin challenge caused down-regulation of adherens junction protein VE-Cadherin in the lung tissues. The down-regulation of VE-Cadherin was also blocked by CAY10603 and Tubastatin A pre-treatment ( Figure 5).

HDAC6 inhibition reduces TNF-α-induced endothelial permeability and attenuates lung edema formation in endotoxemia
TNF-α is a major pro-inflammatory mediator known to induce trans-endothelial hyper-permeability during inflammation [1,7,9]. We examined the effects of HDAC6 inhibition by CAY10603 on TNF-α-induced endothelial permeability. TNF-α challenge caused increased endothelial permeability to FITC-dextran in HPAECs. Cells pre-treated with CAY10603 exhibited significant reduction of TNF-α-induced endothelial permeability to FITC-dextran ( Figure 6A). We also examined the effects of CAY10603 on lung vascular permeability in the mouse model of endotoxemia. In our studies, endotoxin-induced lung edema formation was significantly inhibited by CAY10603 pre-treatment ( Figure 6B).
HDAC6 inhibition has been reported to prolong survival in murine models of systemic inflammation and injury [20,24,25]. However, mechanisms underlying the protective effects observed by HDAC6 inhibition remain to be determined. In the present study, we demonstrated that HDAC6 inhibition potently inhibited TNF-α-induced caspase 3 activation and endothelial barrier dysfunction, suggesting that HDAC6 inhibition could provide protection against inflammation-mediated endothelial cell injury.
We also showed that HDAC6 inhibition effectively suppress caspase-3 activation and maintain lung endothelial barrier integrity in vivo. HDAC6 inhibitors prevented caspase 3 activation in lung tissues with reduced lung edema in a mouse model of endotoxemia. Maintaining VE-Cadherin expression is critical in supporting lung vascular barrier integrity [43]. We demonstrated that Tubastatin A and CAY10603 were able to prevent the reduction of VE-Cadherin in lung tissues after LPS challenge. Our results suggest that HDAC6 inhibition is a new approach that can modulate endothelial cell junction stability in inflammatory lung injury. More studies are needed in the future to further investigate this new pathway.
In summary, selective HDAC6 inhibition by CAY10603 and Tubastatin A prevents TNF-α-induced endothelial cell dysfunction. HDAC6 inhibitors prevent caspase-3 activation in endothelial cells and inhibit TNFα-induced endothelial barrier dysfunction by maintaining cell-cell junction integrity. HDAC6 inhibitors prevented endotoxin-induced lung caspase-3 activation and lung edema, suggesting that selective HDAC6 inhibition possesses therapeutic potential to treat endothelial cell dysfunction during acute inflammation.

Cell culture
Human pulmonary arterial endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) were purchased from Lonza (Allendale, NJ). Cells were grown in EGM-2 supplemented with fetal bovine serum (FBS) and cultured in an incubator at 37°C in 5% CO2 and 95% air. Cells from passages 5 to 9 were used in the experiments.

HDAC6 siRNA knockdown
HDAC6 siRNA knockdown was conducted as described previously [41]. Cells were transfected with siRNAs for 48 h according to the manufacture's protocol. siRNA-transfected cells were then stimulated with 20 ng/ml TNF-α for 24 h.

Immunofluorescence and immunoblotting assays
For immunofluorescence assay, cells were grown on glass coverslips pre-coated with 0.1% gelation. After the treatment, cells were fixed in -20°C methanol and washed with PBS on ice. Cells were blocked with 1% BSA in PBS, then incubated with primary antibody. After 3 washes with TBS, cells were incubated with Alexa Fluor 594 secondary antibody. The coverslips were mounted on the slides with DAPI. Immunoblotting assays of cell and lung tissue samples were conducted as described previously [43,44].

Mouse model of endotoxemia
Eleven to twelve week-old male C57BL/6 mice were purchased from Jackson Lab. All experiments and animal care procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Endotoxemia was induced by I.P. injection of 7.5 mg/kg LPS in phosphate buffered saline (PBS). Mice were pre-treated by I.P. injection with Tubstatin A (9 mg/kg body weight) for 6 h or CAY10603 (5 mg/kg body weight) for 2 h before LPS challenge. Experiments were terminated 24 h after LPS challenge. Lung wet/dry weight ratio was assessed as described previously [43].

Endothelial cell permeability assay
In Vitro Vascular Permeability Assay (96-Well) kit was used to measure endothelial cell monolayer permeability to FITC-dextran according to the manufacture's protocol. Briefly, endothelial cells were seeded into the insert. 72 h later, endothelial cells formed a monolayer. Endothelial cell monolayers were pretreated with CAY10603 (CAY, 0.1 µM) for 6 h, and then stimulated with TNF-α for 18 h. A high molecular weight (2000 kD) FITC-Dextran was diluted in EGM-2 medium (1:40) and added into the insert. 20 minutes after the incubation, 100 µl medium from lower chamber was collected. Fluorescence intensity was measured in a plate with a fluorescence plate reader.

Statistical analysis
Results are expressed as means ± SE of 3 to 6 independent experiments. ANOVA and post hoc multiple comparison tests were used for multiple groups. The Student′s t-test was used for comparisons of two groups. Statistical significance was assigned to P values < 0.05.

ACKNOWLEDGMENTS AND FUNDING
Jinyan Yu is a recipient of the State Scholarship from China Scholarship Council.

CONFLICTS OF INTEREST
All the authors declared no competing interests.