Upregulation of prolylcarboxypeptidase (PRCP) in lipopolysaccharide (LPS) treated endothelium promotes inflammation

Abstract

Background: Prolylcarboxypeptidase (Prcp) gene, along with altered PRCP and kallikrein levels, have been implicated in inflammation pathogenesis. PRCP regulates angiotensin 1-7 (Ang 1-7) - and bradykinin (BK) - stimulated nitric oxide production in endothelial cells. The mechanism through which kallikrein expression is altered during infection is not fully understood. Investigations were performed to determine the association between PRCP and kallikrein levels as a function of the upregulation of PRCP expression and the link between PRCP and inflammation risk in lipopolysaccharide (LPS)-induced endothelium activation.

Methods: The Prcp transcript expression in LPS-induced human umbilical vein endothelial cells (HUVEC) activation was determined by RT-PCR for mRNA. PRCP-dependent kallikrein pathway was determined either by Enzyme Linked ImmunoSorbent Assay (ELISA) or by biochemical assay.

Results: We report that PRCP is critical to the maintenance of the endothelial cells, and its upregulation contributes to the risk of developing inflammation. Significant elevation in kallikrein was seen on LPS-treated HUVECs. The conversion of PK to kallikrein was blocked by the inhibitor of PRCP, suggesting that PRCP might be a risk factor for inflammation.

Conclusion: The increased PRCP lead to a sustained production of bradykinin in endothelium following LPS treatment. This amplification may be an additional mechanism whereby PRCP promotes a sustained inflammatory response. A better appreciation of the role of PRCP in endothelium may contribute to a better understanding of inflammatory vascular disorders and to the development of a novel treatment.

Figure 1

LPS enhances prekallikrein activation and PRCP expression in HUVEC. Panel A: total RNA was isolated from HUVEC and LPS treated HUVEC (LPS-HUVEC) cells and then amplified by RT-PCR. Amplified DNA (100 bp fragment) was resolved on a 1.5% agarose gel. Panel B: 20 nM PK in the absence or presence of inhibitor was incubated with 20 nM HK bound to untreated, LPS-pretreated, or PRCP-siRNA transfected cells pretreated with LPS at 37°C. The liberation of paranitroanilide (pNA) from substrate (S2302) by kallikrein was measured at 405 nm. *p < 0.01 vs. untreated cells. The presented are the mean ± SEM of triplicate points of 10 independent experiments.

Figure 2

PRCP-dependent prekallikrein (PK) activation and bradykinin (BK) liberation on LPS-treated HUVEC. Untreated, LPS-pretreated, or PRCP-siRNA transfected cells pretreated with LPS were incubated with 100 nM HK alone or in the presence of 1 μM HKH20 in HEPES buffer containing 2 mM Ca²⁺ and 1 mM Mg²⁺ at 37°C for 60 min. Afterward, 100 nM PK with 1 μM lisinopril [angiotensin-converting enzyme inhibitor (ACE)] and 1 μM HOE 140 (bradykinin B2 receptor antagonist) was added and incubated with HUVEC at 37°C for 60 min in the same buffer. Forty-eight hours after transfection with 100 nM PRCP-siRNA or control, cells were incubated with HK and PK and assessed for BK generation as described above. After PK activation on HUVEC, the buffer from each of the wells was collected and deproteinized by treatment with trichloroacetic acid. The data are from three experiments (means ± SEM).

Figure 3

Influence of the plasma kallikrein-kinin activation on endothelium monolayer permeability. Endothelial cells (1.0 × 10⁶ cells/ml) were seeded in the inserts of permeability chambers that were coated with collagen. The endothelial cell monolayer were incubated with 300 nM HK, 300 nM PK, or the complex of HK and PK (300 nM each), 300 nM bradykinin, or 0.3 μg/ml LPS for 3 hours at 37°C in the tissue culture incubator. Then, 150 μl of FITC-Dextran (1:30 dilution) was added to each insert and incubated for 5 min at room temperature. The presence of FITC-Dextran (1:30 dilution) in the lower chamber was determined by a Perkinelmer (precisely) Envision 2103 Multimode Reader at excitation wavelength of 485 nm and emission wavelength of 530 nm.

Figure 4

Schematic representation of the PRCP-dependent PK activation pathway on LPS pretreated endothelial cells. PRCP: prolylcarboxypeptidase; PK: prekallikrein; HK: high molecular weight kininogen; BK: bradykinin; FXII: factor XII (Hageman factor); FXIIa: activated factor XII. ↑ Indicates an increase, and the thickness of the arrow illustrates the extent of an increase.