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Unfractionated heparin (UFH) is largely used as anti-thrombotic drug. While UFH has been shown to suppress lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation, intracellular upstream events that cause NF-κB down-regulation in response to UFH remain unclear. Thus, we investigated the involvement of phosphoinositide-3-OH kinase (PI3K)/Akt in the inhibition of LPS-activated NF-κB pathway by UFH in human pulmonary microvascular endothelial cells (HPMECs). Pretreatment with UFH (0.1-1U/ml) significantly inhibited LPS (10μg/ml)-stimulated interleukin (IL)-6 and IL-8 production in HPMECs. LPS activated Akt and NF-κB, whereas UFH suppresses LPS-induced Akt phosphorylation and NF-κB nuclear translocation, which were required for IL-6 and IL-8 gene transcription. Inhibition studies by using wortmannin abrogated NF-κB-mediated IL-6 and IL-8 expression, suggesting the requirement of PI3K/Akt pathway. Our data provided the first evidence that UFH might repress LPS-activated PI3K/Akt pathway, leading to inhibitory effect of NF-κB activation with diminished IL-6 and IL-8 expression in HPMECs.

Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), apart from anticoagulant activities, contain a variety of biological properties such as anti-inflammatory actions possibly affecting sepsis. Chemokines are vital for promoting the movement of circulating leukocytes to the site of infection and are involved in the pathogenesis of sepsis. The purpose of this study was to investigate the effects and potential mechanisms of UFH on lipopolysaccharide (LPS)-induced chemokine production in human pulmonary microvascular endothelial cells (HPMECs). HPMECs were pretreated with UFH (0.1 U/ml and 1 U/ml), 15 min prior to stimulation with LPS (10 μg/ml). Cells were cultured under various experimental conditions for 2 h and 6 h for analysis. UFH markedly decreased LPS-induced interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression in HPMECs. UFH also attenuated the secretion of these chemokines in culture supernatants. In addition, UFH blocked the chemotactic activities of LPS-stimulated HPMECs supernatants on monocytes migration as expected. UFH inhibited LPS-induced Kruppel-like factor 5 (KLF-5) mRNA and protein levels. Concurrently, UFH reduced nuclear factor (NF)-κB nuclear translocation. Importantly, transfection with siRNA targeting KLF-5 reduced NF-κB activation and chemokines expression. These results demonstrate that interfering with KLF-5 mediated NF-κB activation might contribute to the inhibitory effects of chemokines and monocytes migration by UFH in LPS-stimulated HPMECs.