Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury

Abstract

Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS) and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC) apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1) induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control) nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

Fig 1. Microparticles released from monocytic cell THP1 induce human pulmonary microvascular endothelial cell (HPMVEC) death.

THP1 cells were cultured at 5,10,15,20 and 50 million/ml and stimulated with LPS (1μg/ml) for 2h or left untreated. (A) Microparticles (MP) were isolated from each condition and subjected to HPMVEC and analyzed for cell death using MTS assay. (B) MPs were also subjected to careful normalization by total protein prior to subjecting to HPMVEC. (B) Immunoblot analysis of both control MP from 50X10⁶ THP1/ml and LPS MP from 10X10⁶ THP1/ml, normalized based on total protein content was analyzed for β-actin (loading control) and presence of active caspase-1 (p20). (C) Normalized MP fractions (stained with DilC16) were also analyzed by flow cytometry. (D) These MP fractions were then subjected to HMPVEC and analyzed for cell death by MTS assay. (E) LPS amounts were measured from MPs isolated from unstimulated THP1 (50X10⁶ THP1/ml) and LPS stimulated THP1 (10X10⁶ THP1/ml) and nonMP fractions using LAL assay. (F) MPs were also isolated from primary monocytes from Red Cross buffy coats. Monocytes were isolated from buffy coats and stimulated with LPS similar to THP1 cells for 2 and 4 h. Isolated MPs were analyzed for presence of active caspase-1 (p20) and induction of HPMVEC cell death (MPs form 2h stimulation used for MTS assay). Quantitative analysis for n = 2 experiments.

Fig 2. Human pulmonary microvascular endothelial cell apoptosis is mediated by THP-1 stimulated microparticles and inhibited by caspase-1 inhibitor.

THP1 cells were stimulated with LPS (1μg/ml) for 2h and supernatants were separated into microparticle (MP) and non- microparticle (nonMP) fractions by serial centrifugations. HPMVECs were co-cultured with these MP and nonMP fractions and analyzed for cell viability and apoptosis. Cell viability was measured by MTS assay (A and B) and morphology using DAPI staining (C). HPMVECs were then treated with microparticles isolated from unstimulated or LPS induced THP1 in the presence or absence of the caspase-1 inhibitor, YVADcmk (D). Apoptosis was analyzed using Annexin V/PI assay by flow cytometry. Quantitative analysis for n = 3 experiments. * #Comparison of LPS MP to control MP/cells only, $ Comparison of LPS MP to LPS nonMP, @ Comparison of LPS MPs, with and without YVAD.

Fig 3. Active caspase-1 and ASC release in microparticles from stimulated THP-1.

THP1 cells were stimulated with LPS (1μg/ml) for 2h in the presence or absence of YVAD-cmk. Microparticles were isolated from LPS stimulated THP-1 supernatant by stepwise ultracentrifugation (A). The cell fraction was separated by centrifugation at 2000 g for 5 min. Supernatants were then further fractionated into a 16,000g fraction followed by microparticle (MP) fraction and non- microparticle (nonMP) fraction by a final ultracentrifugation at 100,000 g for 1h. Microparticles (MP) and non-microparticle fractions (nMP) were analyzed for presence of the p20 form of caspase-1 (B) and the inflammasome adaptor protein, ASC (C) by immunoblot. Substrate cleavage capacity of caspase-1 from each of the supernatant fraction was also measured using WEHD-enzymatic assay (D). Analysis for n = 2 experiments.

Fig 4. Encapsulation is necessary for exogenous caspase-1 mediated apoptosis.

THP1 cells were first labeled with phospholipid dye, DiLC16 for 30 min, washed, stimulated with LPS (1μg/ml) for 2h and microparticles were isolated. A) Labeled microparticles were observed under fluorescence microscope; left panel (top and bottom). Uptake of labeled microparticles by DAPI stained HPMVEC was observed by fluorescence microscopy (right panel: control MP (top) and LPS MP (bottom)). B) Microparticles were then either kept intact or disrupted by vortexing or by sonication, or subjected to heat inactivation. Microparticles were then applied to HPMVECs and endothelial cell viability was analyzed by MTS assay (n = 3). * intact LPS MP vs control MP or intact LPS MP vs ruptured/ heat inactivated LPS MPs. C) Caspase-1 activity of each fraction was measured using WEHD enzymatic assay from two experiments. Mild homogenization of microparticles or sonication did not affect caspase-1 activity in contrast to heat inactivation. D) HPMVEC were pretreated with cytochalasin D (5μg/ml) for 30 min, washed two times to remove trace of reagent and then control or LPS MP was subjected to cells as previously described. Uptake of microparticles and effect on apoptosis of HPMVEC was analyzed by MTS assay (n = 3 experiments) * intact LPS MP vs control MP; # LPS MP with cytochalasin D vs LPS MP.