Pulmonary midkine inhibition ameliorates sepsis induced lung injury

Jing-Yuan Xu, Wei Chang, Qin Sun, Fei Peng, Yi Yang, Jing-Yuan Xu, Wei Chang, Qin Sun, Fei Peng, Yi Yang

Abstract

Background: Midkine is a multi-functional molecule participating in a various key pathological process. We aimed to evaluate the change of midkine in sepsis and its association with angiotensin-converting enzyme (ACE) system, as well as the mechanism by which midkine induced in sepsis and lung injury.

Methods: The peripheral blood sample of septic patients on admission was obtained and measured for midkine, ACE and angiotensin II. Cecal ligation and puncture (CLP) mouse model was used, and adeno-associated virus (AAV) was stilled trans-trachea for regional targeting midkine expression, comparing the severity of lung injury. Furthermore, we studied the in vitro mechanism of midkine activates ACE system by using inhibitors targeting candidate receptors of midkine, and its effects on the vascular endothelial cells.

Results: Plasma midkine was significantly elevated in sepsis, and was closely associated with ACE system. Both circulating and lung midkine was increased in CLP mouse, and was related to severe lung injury. Regional interfering midkine expression in lung tissue by AAV could alleviate acute lung injury in CLP model. In vitro study elucidated that Notch 2 participated in the activation of ACE system and angiotensin II release, induced by midkine and triggered vascular endothelial injury by angiotensin II induced reactive oxygen species production.

Conclusions: Midkine inhibition ameliorates sepsis induced lung injury, which might via ACE/Ang II pathway and the participation of Notch 2 in the stimulation of ACE. Trial registration Clinicaltrials.gov NCT02605681. Registered 12 November 2015.

Keywords: Angiotensin II; Angiotensin converting enzyme; Midkine; Sepsis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Clinical Profiles. a Plasma midkine remarkedly elevated in septic patients (n = 26) vs. healthy volunteers (n = 5). b The correlation between plasma midkine and ACE (Spearmen’s rho = 0.3793, P = 0.0676). c The correlation between plasma midkine and angiotensin II (Spearmen’s rho = 0.38, P = 0.07)
Fig. 2
Fig. 2
Midkine and ACE level in plasma and lung tissue. a Plasma midkine in CLP (n = 6) vs. sham (n = 6) model [ng/L, 747.3 (703.6–811.1) vs. 87 (64.5–199.5), P = 0.0095]. b Plasma ACE in CLP (n = 6) vs. sham (n = 6) [ng/mL, 454.1 (378.1–500.3) vs. 53.4 (46.3–63.8), P = 0.0022]. c Plasma angiotensin II in CLP (n = 6) vs. sham (n = 6) [ng/L, 7.2 (6.8–9.6) vs. 1.9 (1.2–3.3), P = 0.002]. d Midkine and ACE mRNA level in lung tissue in CLP (n = 6) vs. sham (n = 6), mRNA amplification was normalized to β-actin. e Midkine and ACE protein level was determined by Western blots, and the representative result was shown. Quantitative analysis of midkine and ACE protein using densitometry, normalized to β-actin
Fig. 3
Fig. 3
Targeted abrogation of midkine in the lung tissue ameliorated acute lung injury in CLP model. a Immunofluorescence staining of GFP of lung tissue in control mouse (WT), mouse transduced with vehicle AAV (AAV. Veh) and mouse transduced with AAV carrying midkine RNAi sequence (AAV.RNAi.MK, n = 6 in each group). Lower panel showed immunohistochemistry staining of midkine in lung tissue in WT, AAV.Veh and AAV.RNAi.MK group (n = 6 in each group). b Midkine and ACE protein levels in lung tissue determined by Western blots in control mouse (WT), mouse transduced with vehicle AAV (AAV.Veh) and mouse transduced with AAV carrying midkine RNAi sequence (AAV.RNAi.MK) (n = 6 in each group). c Immunohistochemistry staining of midkine in lung tissue of control mouse (WT) and mouse transduced with AAV midkine RNAi (AAV.RNAi.MK) with sham operation (upper panel), and the respective group mouse with CLP (lower panel) (n = 6 in each group). d H&E staining of control mouse (WT), mouse transduced with vehicle AAV (AAV.Veh) and mouse transduced with AAV carrying midkine RNAi sequence (AAV.RNAi.MK) in CLP vs. sham mouse were shown. Quantitative assessment by lung histopathological score was presented (n = 6). e BALF analysis from control mouse (WT), mouse transduced with vehicle AAV (AAV.Veh) and mouse transduced with AAV carrying midkine RNAi (AAV.RNAi.MK) in CLP vs sham mouse were shown (n = 6). Cell counts (upper left), TNF-α in BALF (upper right), protein contents in BALF (lower left) and lung wet/dry ratio (lower right) were shown (n = 6 in each group). f ACE hydrolytic activity determined by FAPGG in control mouse (WT), mouse transduced with vehicle AAV (AAV.Veh) and mouse transduced with AAV with midkine RNAi (AAV.RNAi.MK) in CLP vs. sham mouse was shown (left). ACE activity was measured at indicated time point. Angiotensin I was added to lung homogenization and the converted angiotensin II was determined (right) (n = 6 in each group)
Fig. 4
Fig. 4
Pulmonary microvascular endothelial cell injury was mediated by ACE system via Notch 2 receptor. a ACE mRNA level in pulmonary microvascular endothelial cell (PMVEC) in control group, cells stimulated by midkine (20 ng/mL and 100 ng/mL), and normalized to β-actin (upper left). ACE protein level in PMVEC lysates in control group, cells stimulated by midkine (20 ng/mL and 100 ng/mL). Quantitative analysis using densitometry normalized to β-actin was shown (lower left). PMVEC ACE activity determined by FAPGG in control group, cells stimulated by midkine, and cell stimulated by midkine and blocked with inhibitors targeting Notch 2 (DAPT), ALK (LDK378) and EGFR (Erlotinib), respectively. ACE activity was measured at indicated time point (upper right). Angiotensin II in the supernatant was determined after angiotensin I was added to the culture medium, in control group, cells stimulated with midkine, and cells stimulated with midkine blocked by DAPT (lower right). b Notch 2 and ACE protein level in PMVEC in control group, cells stimulated with midkine (100 ng/mL), and cells stimulated by midkine and blocked by DAPT. Quantitative analysis of full length (FL) and intra-cellular domain (NICD) of Notch 2 and ACE, using densitometry, normalized to β-actin. c Cell viability determined by CCK-8 assay of PMVEC in blank, control group, cells stimulated by midkine, cells stimulated by midkine and blocked by DAPT and ACEI respectively. Reactive oxygen species (ROS) production in PMVEC in blank, control group, cells stimulated by midkine, cells stimulated by midkine and blocked by DAPT and ACEI respectively. d The expression of inter-cellular adhesive molecules including VCAM-1 and VE-cadherin were determined by Western blots. The expression of VCAM-1 and VE-cadherin in PMVEC were shown in control group, cells stimulated by midkine, cells stimulated by midkine and blocked by DAPT and ACEI, respectively. e Immunofluorescence staining of VE-Cadherin and ZO-1 in PMVEC in control group, cells stimulated by midkine and cells stimulated by midkine but prohibited by DAPT

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