Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor superfamily, and is the main physiologic inhibitor of urokinase plasminogen activator (u-PA) and tissue-type plasminogen activator . Studies using PAI-1-deficient (PAI-1−/−) mice have demonstrated that PAI-1 not only regulates the fibrinolytic system, but also modulates other physiologic and pathophysiologic processes, including inflammation, angiogenesis, tumor growth, and cardiovascular disease [2–6]. Regions of human PAI-1 that are critical for inhibition of plasminogen activation, binding to vitronectin (VN) and binding to LDL receptor-related protein have been identified [7–9]. Our laboratory has demonstrated that these functional sites are conserved in the murine system . In order to define functional roles for domains within PAI-1, we generated mice that express PAI-1 with altered VN-binding capacity. Binding of VN to PAI-1 stabilizes the biological activity of PAI-1 and prolongs its half-life in plasma [11–13]. Moreover, the binding of VN by PAI-1 modulates cell adhesion and cell migration by limiting the binding of VN to its integrin receptor and u-PA receptor (uPAR) [13–17].
It has been demonstrated that a Q123K mutation in PAI-1 results in a significant reduction in the ability of PAI-1 to bind to VN , and transgenic mice overexpressing this PAI-1 variant have been generated and characterized . Previous studies have shown that a recombinant murine PAI-1 variant that has the double mutation R101A/Q123K has significantly diminished ability to bind to VN, even more so than that with the single mutation at amino acid 123 . On the basis of these findings, genetic mutations that translate into these alterations (R101A and Q123K) of PAI-1 were targeted into the PAI-1 gene in the mouse genome. A 2806-bp PCR genomic fragment containing PAI-1 exon 2 and exon 3 was subcloned into the pCR.21-TOPO vector as the 5′-flank for the targeting vector (TV), and nucleotide substitutions were introduced by site-directed mutagenesis to generate the R101A and Q123K changes in exon 3. A 3674-bp PCR genomic fragment containing PAI-1 exons 4 and 5 was subcloned into the pCR.21-TOPO vector as the 3′-flank for the TV. The 5′-flank and 3′-flank were cloned into the multicloning site of a pre-made TV backbone in which the neomycin resistance gene (Neo) cassette (NEO) was flanked by two lox P (Lox) sites and two flippase recombination target (FRT) sites to yield the final TV for PAI-1 VN with the R101A and Q123K mutations (Fig. 1A).
The TV was electroporated into C57BL/6/129 embryonic stem (ES) cells. The ES cells surviving negative selection with 5′-fluorocytosine for the cytosine deaminase cassette gene and positive selection with G418 for the NEO gene were screened by southern blot analysis, and the mutations were confirmed by PCR (data not shown). A PCR strategy was also employed to confirm homologous recombination (Fig. 1B). Recombined ES cells were injected into blastocysts, and chimeric males were identified. The F1 offspring resulting from crossing chimeric male mice with C57BL/6 female mice were tested for proper germline transmission by PCR and sequence analysis (Fig. 1B). F1 mice were then bred with transgenic mice expressing flippase, Tg-CAG_FLPe37, to remove the NEO gene (Fig. 1B). The PCR forward primer 5′-GCTCAACATGAGCCTAATGGATC-3′ and reverse primer 5′-CATTCATGAGTTCCTGGCTCCAG-3′ were used to detect the removal of the NEO gene. A PAI-1 genomic fragment from PAI-1R101A/Q123K mice was cloned and sequenced, and it was found to contain the mutations for R101A and Q123K, and the FLPe/FRT recombination sequence. Blood counts and blood analyses were performed, and body weights and litter sizes were determined, for wild-type (WT), PAI-1−/− and PAI-1R101A/Q123K mice, and all values were within the normal range.
Lipopolysaccharide (LPS) is derived from the outer membrane of Gram-negative bacteria, and is a known inducer of PAI-1 gene expression . In order to determine whether this response is equivalent between WT and PAI-1R101A/Q123K mice, LPS (2 μg g−1 body weight, E.C. 0111:B4; Sigma-Aldrich Co. LLC, St. Louis, Mo) was injected intraperitoneally into 8–12-week-old male WT and PAI-1R101A/Q123K mice. After 8 h, plasma PAI-1 levels and PAI-1 inhibitory activity were determined. Plasma levels of PAI-1 in WT and PAI-1R101A/Q123K mice were equivalent (Fig. 1Ca), as was plasma PAI-1 inhibitory activity (Fig. 1Cb). In addition, the mRNA levels of PAI-1 in liver were also the same for the two genotypes (Fig. 1C-c). These results indicated that PAI-1R101A/Q123K mice have the same ability to produce PAI-1 as WT mice, and that the PAI-1 from PAI-1R101A/Q123K mice maintained plasminogen activator inhibitory activity. LPS is the major causative agent in Gram-negative endotoxemia. Hallmark features of this disease are a systemic inflammatory response and hypercoagulablility. The systemic microthrombosis that develops leads to disseminated intravascular coagulation, and subsequently to hypoxic organ failure [21–23]. Indeed, our laboratory has previously shown that a coagulation factor VII deficiency protects against lethal endotoxemia . Thus, a balance between the fibrinolytic system and the coagulation system plays an important role in regulating the downstream effects of endotoxemia. As PAI-1 is a target following LPS exposure, it was determined whether a lack or mutation of PAI-1 would affect survival after exposure to a lethal dose of LPS. For this study, 8–12-week-old male WT, PAI-1−/− and PAI-1R101A/Q123K mice were injected intraperitoneally with LPS (10 μg g−1 body weight), and survival was monitored every 3 h. PAI-1−/− and PAI-1R101A/Q123K mice were shown to have a significant (P = 0.03 and P = 0.011, respectively) survival advantage relative to WT mice (Fig. 1D). Studies have demonstrated that VN levels in lungs following intratracheal administration of LPS are significantly increased . With this model, VN null mice were protected against LPS-induced acute lung injury . Additionally, other studies have demonstrated that there is an increased occurrence of neutrophil apoptosis in LPS-treated VN null mice , and apoptotic cells can protect mice from LPS toxicity . In the current study, PAI-1 played a critical role in LPS-induced lethality, and the VN-binding capacity of PAI-1 is important for this function, potentially through the enhanced effect of VN on PAI-1 inhibition of the anti-coagulant, anti-inflammatory protein, protein C .