Human Urinary Kallidinogenase Promotes Angiogenesis and Cerebral Perfusion in Experimental Stroke

Abstract

Angiogenesisis a key restorative mechanism in response to ischemia, and pro-angiogenic therapy could be beneficial in stroke. Accumulating experimental and clinical evidence suggest that human urinary kallidinogenase (HUK) improves stroke outcome, but the underlying mechanisms are not clear. The aim of current study was to verify roles of HUK in post-ischemic angiogenesis and identify relevant mediators. In rat middle cerebral artery occlusion (MCAO) model, we confirmed that HUK treatment could improve stroke outcome, indicated by reduced infarct size and improved neurological function. Notably, the 18F-FDG micro-PET scan indicated that HUK enhanced cerebral perfusion in rats after MCAO treatment. In addition, HUK promotespost-ischemic angiogenesis, with increased vessel density as well as up-regulated VEGF andapelin/APJ expression in HUK-treated MCAO mice. In endothelial cell cultures, induction of VEGF and apelin/APJ expression, and ERK1/2 phosphorylation by HUK was further confirmed. These changes were abrogated by U0126, a selective ERK1/2 inhibitor. Moreover, F13A, a competitive antagonist of APJ receptor, significantly suppressed HUK-induced VEGF expression. Furthermore, angiogenic functions of HUK were inhibited in the presence of selective bradykinin B1 or B2 receptor antagonist both in vitro and in vivo. Our findings indicate that HUK treatment promotes post-ischemic angiogenesis and cerebral perfusion via activation of bradykinin B1 and B2 receptors, which is potentially due to enhancement expression of VEGF and apelin/APJ in ERK1/2 dependent way.

Fig 1. HUK protected against ischemic brain injury.

(A) Representative TTC images showing infarct size 3 d after MCAO. The normal tissue was stained deep red and the infarct area was stained pale gray. (B) Statistic bar graph of brain infarct size in vehicle or HUK-treated rats at 1 d, 3 d, and 7 d after MCAO. **p<0.01 versus vehicle group, N = 6. (C) Neurological performance of rats in each group assessed with longa score at 3 h, 1 d, 3 d, 7 d, and 14 d after reperfusion. *p<0.05 versus control group, N = 10 per group.

Fig 2. HUK enhanced cerebral blood perfusion and promoted angiogenesis in stroke rats.

(A) Representative images of ¹⁸F-FDG PET/CT in HUK group and vehicle group at 1 d and 14 d after stroke. (B) Uptake of ¹⁸F-FDG in HUK or vehicle-treated rats at different time points after stroke. *p<0.05 versus vehicle group; **p<0.01 versus vehicle group, N = 10 per group. (C) Left panel showed the typical detected areas of CD31 staining in rats brain indicated in boxed area. Right panel demonstrated representativeimmunofluorescent images stained by CD31 (green) 7 days after reperfusion. Scale bar = 100 μm. (D) CD31 positive vessels were counted and analyzed at indicated time point. Data was presented as capillary density reflected by number of CD31 positive vessels per mm². #p<0.05 versus sham group; ##p<0.01 versus sham group; *p<0.05 versus vehicle group; **p<0.01 versus vehicle group. N = 6 per group. (E) Quantitative data of CD31 mRNA levels at 1, 3, 7, and 14 d after stroke onset. # p<0.05 versus sham group; *p<0.05 versus control group. N = 6 per group.

Fig 3. HUK enhanced VEGF, Apelin/APJ expression in stroke rat brain.

MCAO rats with or without HUK treatment were sacrificed and mRNA was extracted for the detection of apelin (A), APJ (B), VEGF (C) levels by real-time PCR. N = 6 per group. #p<0.05 versus sham group; ##p<0.01 versus sham group; *p<0.05 versus control group; **p<0.01 versus control group. Protein level of apelin in rat brain was determined by ELISA (F), while protein level of VEGF in rat brain was measured by Western blot (D). Quantitative data of VEGF blots at indicated time points (F). #p<0.05 versus sham group; *p<0.05 versus vehicle group, N = 6 repeats.

Fig 4. HUK up-regulated pro-angiogeic factors in ERK1/2 dependent way in cultured endothelial cells.

mRNA from cultured HAECs treated with HUK only or HUK plus U0126 was isolated for the measurement of VEGF (A), apelin (B), and APJ (C). N = 6 per group. (D) Western blot showed the activation of ERK pathway and VEGF expression in cultured HAECs incubated with HUK only or U0126 plus HUK for 15 min, 1 h, 3 h and 24 h. Statistic bar graph of p-ERK/ERK blots (E) and VEGF blots (F), N = 6 per group. (G) ELISA was used to measure apelin concentration in cultured cell medium. (H) F13A decreased VEGF expression up- regulated by HUK. (I) Quantitative data of VEGF blots intensity with or without F13A. #p<0.05 versus control group *p<0.05 versus HUK group; **p<0.01 versus HUK group; ##p<0.01 versus control group. N = 6 per group.

Fig 5. HUK promoted angiogenesis through activation of bradykinin B1 and B2 receptors.

(A) Representative images of CD31 immunostaining (green) in rats peri-infarct cortex 7 days after reperfusion. Both R715 (0.5mg/kg) and HOE140 (100μg/kg) reduced capillary density induced by HUK in stroke rats. Scale bar in upper panel = 100 μm, in lower panel = 50 μm. (B) Capillary density was quantified by CD31 positive vessels per mm². (C) Representative images of tube formation assay. 30 minutes pre-treatment with R715 (0.5 μM) orHOE140 (1 μM) inhibited HUK-induced increase in tube formation capacity of OGD-treated HAECs. Scale bar = 100 μm (D) Total tube length of capillary like structure was analyzed and presented as total tube length permm2. (E) Real-time PCR demonstrated that blockade of either B1 or B2 receptors inhibited HUK-induced increase in mRNA levels of VEGF, apelin, and APJ of OGD-treated HAECs. (F) Upper panel showed VEGF protein expression detected by western blot. Lower panel demonstrated quantification of VEGF blots intensity using Image J software. Protein expression of VEGF was normalized to internal control, β-tubulin. Blockade of either B1 or B2 receptors inhibited HUK-induced VEGF expression at protein levels of OGD-treated HAECs. *** p<0.001 versus Sham group, **p<0.01 versus control group, *p<0.05 versus vehicle group; #p<0.05 versus HUK group. N = 6 per group.