High molecular weight kininogen activates B2 receptor signaling pathway in human vascular endothelial cells

Abstract

The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.

FIGURE 1.

Effect of HK on [Ca²⁺]_i in endothelial cells. Panel A, HK increases [Ca²⁺]_i in HPAEC in a dose-dependent manner. HPAEC grown on coverslips were incubated with 1 μm fluo-4 AM, mounted on a perfusion chamber and continuously perfused with HEPES buffer at the rate of 1 ml/min. Cells were treated with increasing concentrations (100, 300, and 1000 nm) of HK for 3 min each with 5 min wash with HEPES buffer in between each concentration to allow recovery of basal [Ca²⁺]_i. Changes in [Ca²⁺]_i levels in cells were measured with a Zeiss LSM 510 META confocal microscope at an excitation wavelength of 488 nm and an emission wavelength of 505 nm. Change in fluorescence in a field of 10 to 30 endothelial cells was measured. The percentage of endothelial cells which responded to HK stimulation was also determined (inset). Panel B, comparison of the effect of HK, BK, and HK+BK on [Ca²⁺]_i levels in HPAEC. HPAEC were perfused with HK (300 nm), BK (300 nm), or HK+BK (300 nm each). The increase in [Ca²⁺]_i was measured in each cell and expressed as area under curve. Data are presented as mean ± S.E. The changes in [Ca²⁺]_i expressed as change in fluorescence in response to HK or BK in representative endothelial cells are shown (inset). Panel C, characterization of HK by SDS-PAGE. SDS-PAGE was performed with pure HK (300 nm), PK (300 nm), and 2-HK (two chain BK free HK, 300 nm) as well as HK (300 nm), and 2-HK (300 nm) incubated with HPAEC. HPAEC cultured in 96-well microtiter plate were blocked with 1% gelatin for 1 h at 37 °C. Cells were then washed and incubated with HK or 2-HK in the presence of lisinopril (1 μm). After 1 h of incubation at 37 °C, cell supernatant was collected for SDS-PAGE. Electrophoresis was carried out using 4–12% Bis-Tris gel and MES running buffer under non-reducing conditions. After electrophoresis, protein bands were detected using silver staining. Panel D, Western blot analysis of HK metabolism on HPAEC. Electrophoresis was performed as described in panel C on cell supernatants collected after incubating HPAEC with HEPES, lisinopril (1 μm), HK (300 nm)+lisinopril, or 2-HK (300 nm)+lisinopril for 1 h at 37 °C. After electrophoresis, proteins were transferred onto nitrocellulose membrane at 100 V for 1 h. Western blotting was performed using mouse anti-human HK antibody (1:500) and HRP-labeled goat anti-mouse IgG (1:1000) as described under “Experimental Procedures.” Panel E, characterization of HK on HPAEC by LC/MS analysis. HPAEC (10⁴ cells/well) were plated and cultured in 96-well plates. Triplicate samples of cells were incubated with HK (600 nm), PK (600 nm) and HK+PK (600 nm each). The generation of BK in the presence of HOE 140 (1 μm) and lisinopril (1 μm) was determined by LC/MS.

FIGURE 2.

HK stimulates the mobilization of IP₃-sensitive intracellular Ca²⁺ pool as well as extracellular Ca²⁺ influx in HPAEC. HPAEC were treated with HK (300 nm) in the presence or absence of inhibitors. Changes in [Ca²⁺]_i levels in cells were measured with a Zeiss LSM 510 META confocal microscope at an excitation wavelength of 488 nm and an emission wavelength of 505 nm, and the data expressed in terms of area under curve. Panel A, effect of thapsigargin (TG, 2 μm) on HK-induced increase in [Ca²⁺]_i. HPAEC were perfused with TG in a Ca²⁺-free buffer containing EGTA (1 mm) to deplete the intracellular Ca²⁺ stores. Cells were then perfused with HK (300 nm). Changes in [Ca²⁺]_i were measured using confocal microscopy and expressed as area under curve. Data are presented as mean ± S.E. Panel B, effect of EGTA on HK-induced increase in [Ca²⁺]_i. HPAEC were perfused with either HEPES buffer without EGTA or calcium-free buffer with EGTA (1 mm) throughout the experiment. Cells were then treated with HK (300 nm) or HK+BK (300 nm each, control). Changes in [Ca²⁺]_i were measured in each cell using a dual excitation digital Ca²⁺ imaging system. Changes in [Ca²⁺]_i are expressed as ΔF₃₄₀/F_380. Data are presented as mean ± S.E. Panels C–E, effect of gadolinum (Gd³⁺, 10 μm) (C), nifedipine (10 μm) and verapamil (20 μm) (D) and quinine (1 μm) and apamin (1 μm) (E) on HK-induced increase in [Ca²⁺]_i. The increase in [Ca²⁺]_i was measured and expressed as area under curve. Data are presented as mean ± S.E. Panel F, effect of hypertonic stimulation of HPAEC on HK-induced increase in [Ca²⁺]_i. Because Ca²⁺-activated channels are markedly suppressed by hypertonic stimulation of cells, the osmolarity of cell perfusing medium (300 mOsm/liter) was increased by adding 100 mm NaCl (200 mOsm/liter). Cells were then treated with HK (300 nm) or BK (300 nm) and the changes in [Ca²⁺]_i were measured. ***, p < 0.001. *, p < 0.05.

FIGURE 3.

HK activates bradykinin B₂ receptors on HPAEC. Panel A, effect of HOE140 on HK-induced increase in [Ca²⁺]_i. Panel A, HPAEC were treated with HK (300 nm) in the absence or presence of HOE140 (1 μm). Changes in [Ca²⁺]_i levels in cells were measured using a Zeiss LSM 510 META confocal microscope at an excitation wavelength of 488 nm and an emission wavelength of 505 nm and the expressed as area under curve. Data are presented as mean ± S.E. Panel B, effect of U73122 and 2-APB on HK-induced increase in [Ca²⁺]_i. HPAEC were treated with HK (300 nm) in the absence or presence of U73122 (5 μm) or 2-APB (100 μm). Changes in [Ca²⁺]_i levels in cells were measured as described in panel A. Data are presented as mean ± S.E. Panel C, effect of HKH20, HOE140 and BK on biotin-HK binding to HPAEC. HPAEC (4 × 10⁴ cells/well) were incubated with biotin-HK (20 nm) in the absence or presence of increasing concentration of HKH20 (0.1–30 μm) or HOE140 (0.3–100 μm) for 1 h at 37 °C. The binding of biotin-HK to cells was determined using ImmunoPure streptavidin horseradish peroxidase conjugate and peroxide specific fast-reacting substrate turbo-TMB. The reaction was stopped by adding 1 m phosphoric acid (100 μl) and the level of binding was determined by measuring the absorbance of the reaction mixture in each well at OD 450 nm. Inset, effect of BK and HKH20 + BK on biotin-HK binding to HPAEC. HPAEC (4 × 10⁴ cells/well) were treated with biotin-HK (20 nm) in the absence or presence of HKH20 (0.3 μm), BK (0.5 mm), or HKH20 (0.3 μm) + BK (0.5 mm) and incubated for 1 h at 37 °C. Biotin-HK binding was determined as described in panel C. *, p < 0.05; ***, p < 0.001.

FIGURE 4.

Effect of HK on NO and prostacyclin (PGI₂) production and endothelial permeability. Panel A, HK induces NO generation in HPAEC. HPAEC were treated with 100 nm HK alone or with anti-B₂ receptor antibody (1:500), HOE-140 (1 μm), U73122 (100 μm), or 2-APB (100 μm) and incubated for 1 h at 37 °C. The amount of nitrate + nitrite in each sample was measured using a fluorometric assay. Data are presented as mean ± S.E. Panel B, HK stimulates PGI₂ production in HPAEC. HPAEC were incubated with HK (300 nm), bradykinin-free HK (BK-free HK, 300 nm), or HK (300 nm) plus HOE 140 (1 μm) for 1 h at 37 °C. The samples from each well were collected to measure the amount of 6-keto PGF_1α using a competitive AChE enzyme immunoassay. The amount of 6-keto PGF_1α was determined my measuring the change in absorbance at 405 nm using BioTek ELx800 Microplate Reader. Data are presented as mean ± S.E. Panel C, HK influences endothelial permeability. HPAEC monolayer grown on collagen-coated inserts was treated with cell basal medium (negative control); 1 μg/ml LPS (positive control); 0.3 μm and 1 μm HK in the presence or absence of HOE-140 (1 μm); 0.3 μm and 1 μm BK-free HK and incubated at 37 °C for 18 h. The effect on endothelial permeability was determined by adding 150 μl of 1:20 FITC-Dextran to each insert and incubating for 5 min at room temperature.100 μl of solution from each well was then transferred to a 96-well Greiner Microlon 200 Black fluorescence detection plate and the fluorescence read at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Data are presented as mean ± S.E.

FIGURE 5.

Proposed mechanisms of HK-mediated Ca²⁺ signaling in endothelial cells. The binding of HK (domains 3 and 5) to cytokeratin 1 (CK1), urokinase plasminogen activator receptor (uPAR) and complement C1q receptor (gC1qR) on endothelial cells is well established. Our novel findings suggest that in addition to these proteins, HK (through its BK sequence in domain 4) directly interacts with BK B2 (B₂) receptors to regulate endothelial cell functions. HK-mediated stimulation of G_q-coupled B₂ receptors on endothelial cells triggers the activation of PLC-IP3-DAG second messenger signaling pathway. IP3 acts on IP3 receptors (IP3-R) on the endoplasmic reticular (ER) membrane leading to mobilization of intracellular Ca²⁺ from the IP3-sensitive Ca²⁺ pool. An increase in [Ca²⁺]_i activates eNOS and phospholipase A2 (PLA2) to NO and PGI₂ production, respectively. The known inhibitors of B₂ receptor signaling pathway block NO and PGI₂ formation by uncoupling the upstream signaling steps involved in the synthesis of these mediators. Endothelial cell activation in response to HK also involves extracellular Ca²⁺ influx into cells, mainly through B₂ receptor-operated (ROC) and/or store-operated (SOC) Ca²⁺ channels. Gd³⁺, a stretch-activated Ca²⁺ channel inhibitor and verapamil, a L-type voltage gated Ca²⁺ channel inhibitor, potentiate the HK-mediated increase in [Ca²⁺]_i through a yet unidentified mechanism (?). The increase in [Ca²⁺]_i in response to HK activates Ca²⁺-activated Cl⁻ channels (Cl_Ca), and the inhibition of these channels is associated with a decrease in HK-induced rise in [Ca²⁺]_i.