Downregulation of CD151 restricts VCAM-1 mediated leukocyte infiltration to reduce neurobiological injuries after experimental stroke

Abstract

Background: Translational failures in anti-adhesion molecule therapies after stroke reveal the necessity of developing new strategies that not only interrupt leukocyte recruitment but also consider the inhibition of endothelial cell inflammation, verification of therapeutic time window, and normal function maintenance of circulating leukocytes. Our study focused on the potential therapeutic value of CD151 downregulation in improving current anti-adhesion molecule therapies.

Methods: Lentivirus intracerebroventricular administration was conducted to inhibit the CD151 expression and observe its functional influence on neurological injuries and outcomes. Then, immunohistochemistry and myeloperoxidase activity assessment were performed to explore the effects of CD151 expression on neutrophil and monocyte recruitment after rat cerebral ischemia. Primary rat brain microvascular endothelial cells were subjected to oxygen glucose deprivation and reoxygenation to elucidate the underlying working mechanisms between CD151 and VCAM-1.

Results: The CD151 downregulation remarkably reduced neurological injuries and improved neurological outcomes, which were accompanied with reduced neutrophil and monocyte infiltration after the CD151 downregulation. The VCAM-1 expression was remarkably decreased among the adhesion molecules on the endothelial cell responsible for neutrophil and monocyte infiltration. The activation of p38 MAPK and NF-κB pathways was restricted after the CD151 downregulation. p38 MAPK and NF-κB inhibitors decreased the VCAM-1 expression, and p38 acted as an upstream regulator of NF-κB. However, CD151 downregulation did not directly influence the neutrophil and monocyte activation.

Conclusions: Overall, CD151 regulated the expression of adhesion molecules. It also played a critical role in suppressing VCAM-1-mediated neutrophil and monocyte infiltration via the p38/NF-κB pathway. This study possibly provided a new basis for improving current anti-adhesion molecule therapies.

Keywords: CD151; Endothelial cell; Monocyte; Neutrophil; Stroke; VCAM-1.

Fig. 1

In vivo experimental results suggested that the CD151 downregulation had a protective function after experimental stroke. a, b In vivo experiment design. Rats were subjected to LV shCD151/LV vehicle subventricular injection 7 days prior to 2-h middle cerebral artery occlusion ischemia surgery. After 24- or 72-h reperfusion, rats underwent a sacrifice or b behavioral test. c Successful lentivirus transfection was detected by GFP expression in brain cryosections both in the LV vehicle and LV CD151 shRNA groups; the white dotted line showed the right lateral ventricles (scale bar = 500 μm). d Rat’s brain tissue for infarct volume (TTC), brain water content (BWC), and Evan’s blue (EB) measurements. e Infarction volume measured using TTC (n = 6 per group). ** vs. LV Vehicle + MCAO indicates p < 0.01. f BWC 24 h after reperfusion (n = 6 per group, no significant difference was found). g EB test for the evaluation of BBB degradation (n = 6 per group). *** vs. LV Vehicle + MCAO indicates p < 0.001. h Neuronal apoptosis at 24 h after reperfusion (n = 3 per group, scale bar = 100 μm). ** vs. LV Vehicle + MCAO indicate p < 0.01. i Neurobehavioral tests, including Longa’s score, Bederson score, forelimb placing test, beam walk test, 24 h and 72 h after reperfusion (n = 6 per group). *, **, and *** vs. LV Vehicle + MCAO indicate p < 0.05, 0.01, and 0.001, respectively

Fig. 2

CD151 downregulation alleviated the neutrophil and monocyte infiltration after ischemic stroke. a Neutrophil infiltration with a zoomed version (scale bar = 50 μm). Black arrows indicated MPO-positive cells. b Monocyte infiltration with a zoomed version (scale bar = 50 μm). Black arrows indicated CD115-positive cells. c Neutrophil infiltration decreased 24 and 72 h after reperfusion in vivo (n = 3 per group). ** vs. LV Vehicle + MCAO indicates p < 0.01. d The MPO activity, which was used as a neutrophil activation indicator, was reduced in the LV CD151 shRNA + MCAO group 24 and 72 h after reperfusion in vivo (n = 6 per group). ** and **** vs. LV Vehicle + MCAO indicate p < 0.01 and 0.0001, respectively. e Monocyte infiltration decreased 24 and 72 h after reperfusion in vivo (n = 3 per group). ** vs. LV Vehicle + MCAO indicates p < 0.01

Fig. 3

CD151 downregulation influenced the VCAM-1 expression in vivo. a VCAM-1, b ICAM-1, c E-selectin, and d CD9 expression levels evaluated using WB in vivo (n = 6 per group). * and ** vs. LV Vehicle + MCAO indicates p < 0.05 and p < 0.01, respectively. & vs. LV Vehicle + sham indicates p < 0.05

Fig. 4

CD151 downregulation influenced the VCAM-1 expression in vitro. a In vitro experiment design. BMVECs underwent 2 h OGD and 24 h reoxygenation and collected for protein extraction and tests. b VCAM-1, c ICAM-1, d E- selectin, and e CD9 expression levels evaluated using WB in vitro (n = 3 per group). **** vs. LV Vehicle + OGD indicates p < 0.0001. &&&& vs. LV vehicle indicates p < 0.0001

Fig. 5

The MAPK and the NF-κB pathways were inhibited after the CD151 downregulation in endothelial cells. The MAPK kinase (i.e., p38, JNK, and ERK) activation was evaluated using a–c brain tissue (n = 6 per group) and g–i in vitro (n = 3 per group) experiments. ** vs. LV Vehicle + MCAO indicates p < 0.01. The NF-κB pathway activation evaluated using the d, j IκB α degeneration and the p65 translocation from the e, k cytoplasm to the f, l nucleus in enriched endothelial cells (n = 3 per group) and in vitro (n = 3 per group). * and ** vs. LV Vehicle + MCAO indicate p < 0.05 and 0.01, respectively

Fig. 6

CD151 downregulation influenced the VCAM-1 expression through the p38/NF-κB pathway. a In vitro experiment design with inhibitor treatment. SB203580 (p38 inhibitor, 30 μM), SP600125 (JNK inhibitor, 30 μM), PD98059 (ERK 1/2 inhibitor, 30 μM), and PDTC (NF-κB inhibitor, 50 μM) were applied in the LV Vehicle + OGD group. b VCAM-1, c ICAM-1, d E-selectin, and e CD9 expression levels after inhibitor application (n = 3 per group). *, ***, and **** vs. LV Vehicle + OGD indicate p < 0.05, 0.001, and 0.0001, respectively. IκB α degeneration (f) and p65 translocation from the g cytoplasm to the h nucleus were used to evaluate the NF-κB activation (n = 3 per group). * and ** vs. LV Vehicle + OGD indicate p < 0.05 and 0.01, respectively

Fig. 7

No direct effect was detected in the activation of neutrophil and monocyte after stimulation by the BMVEC supernatant. a, b In vitro experiment design: the collected BMVEC supernatant was added into the HL-60 cell line or THP-1 culture media and cultured for 72 h. b ROS production in HL-60 was tested. *** vs. LV Vehicle + OGD indicate p < 0.001. c NET formation in HL-60 was detected by ELISA. **** vs. LV Vehicle + OGD indicate p < 0.0001. d ROS production in THP-1 was tested. ** and *** vs. LV Vehicle + OGD indicate p < 0.01 and 0.001, respectively. e sCD14 secretion in THP-1 supernatant was detected by ELISA. * vs. LV Vehicle + OGD indicate p < 0.05. f sCD163 secretion in THP-1 supernatant was detected by ELISA. *** vs. LV Vehicle + OGD indicate p < 0.001