Serp-1 is a secreted myxoma viral serine protease inhibitor (serpin) with proven, highly effective, anti-inflammatory defensive activity during host cell infection, as well as potent immunomodulatory activity in a wide range of animal disease models. Serp-1 binds urokinase-type plasminogen activator (uPA) and the tissue-type PA, plasmin, and factor Xa, requiring uPA receptor (uPAR) for anti-inflammatory activity. To define Serp-1-mediated effects on inflammatory cell activation, we examined the association of Serp-1 with monocytes and T cells, effects on cellular migration, and the role of uPAR-linked integrins and actin-binding proteins in Serp-1 cellular responses. Our results show that Serp-1 associates directly with activated monocytes and T lymphocytes, in part through interaction with uPAR (P<0.001). Serp-1, but not mammalian serpin PA inhibitor-1 (PAI-1), attenuated cellular adhesion to the extracellular matrix. Serp-1 and PAI-1 reduced human monocyte and T cell adhesion (P<0.001) and migration across endothelial monolayers in vitro (P<0.001) and into mouse ascites in vivo (P<0.001). Serp-1 and an inactive Serp-1 mutant Serp-1(SAA) bound equally to human monocytes and T cells, but a highly proinflammatory mutant, Serp-1(Ala(6)), bound less well to monocytes. Serp-1 treatment of monocytes increased expression of filamin B actin-binding protein and reduced CD18 (beta-integrin) expression (P<0.001) in a uPAR-dependent response. Filamin colocalized and co-immunoprecipitated with uPAR, and short interference RNA knock-down of filamin blocked Serp-1 inhibition of monocyte adhesion. We report here that the highly potent, anti-inflammatory activity of Serp-1 is mediated through modification of uPAR-linked beta-integrin and filamin in monocytes, identifying this interaction as a central regulatory axis for inflammation.

Serpins (serine protease inhibitors) are ubiquitous, complex, and highly active regulatory molecules that effectively control multiple coagulation, inflammatory, and neuroendocrine pathways (1-3). The amino acid sequence in the reactive center loop (RCL) 3 of serpins acts as bait for target serine proteases, initiating structural changes in the serpin-protease complex and culminating in suicide inhibition (1-3). This same RCL can insert into the neighboring ␤-sheet A in other serpins in serpinopathies, causing serpin aggregates induced by genetic mutations and causing disease as for anti-thrombin III (SERPIN C1, ATIII), ␣-1 antitrypsin (SERPIN A1, AAT), and neuroserpin (SERPIN I1, NSP). Whereas the amino acid residues in the RCL provide target P1-P1Ј sequences, referred to as a scissile bond, serpins also require the greater part of the protein structure to function with true serpin-protease inhibitory activity (4, 5). However, as for other proteins, peptides derived during protein metabolism may act to extend serpin activity beyond the initial suicide-inhibitory function, both increasing and decreasing responses (6 -9). In prior work, significant and prolonged antiinflammatory functions have been detected with myxomavirus-derived Serp-1 (10 -17) and mammalian serpin NSP purified protein injections in animal models of vascular disease (18,19). We have hypothesized that peptides produced by protease cleavage of the RCL sequence during natural proteolytic metabolism of Serp-1 or NSP may extend serpin activity, increasing anti-inflammatory activity after serpin-protease complex formation. Thus, these serpin RCL peptide metabolites have the theoretical potential to interfere with either protease activity by acting as a protease bait or inhibitors or to inhibit other serpins by inserting into the ␤-sheet.Many proteins have active metabolites providing additional and/or expanded functions. Peptides derived from calreticulin (20) and apolipoprotein E (Ep1B) (21) have proven anti-atherogenic activity, reducing inflammation and plaque growth in animal models. Serpins also have reported active terminal peptide metabolites. Among the serpins, angiotensinogen is a protein with serpin structure but lacking serpin inhibitory activity (6).

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Thrombolytic serine proteases not only initiate fibrinolysis, but also are up-regulated in vascular disease and acute inflammatory responses. Although the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is considered a main regulator of thrombolysis, PAI-1 is also associated with vascular inflammation. The role of other serpins that target thrombolytic proteases, PAI-2, PAI-3, and neuroserpin (NSP), in vascular inflammation is, however, less well defined. NSP is a mammalian serpin that, similar to PAI-1, inhibits urokinase- and tissue-type plasminogen activators (uPA and tPA, respectively) and has been most closely associated with the nervous system, with a demonstrated protective role after cerebral infarction in mouse models. However, the role of NSP in systemic arterial inflammation and plaque growth is not known. Serp-1 is a myxoma viral serpin that also inhibits tPA and uPA, as well as additionally inhibiting plasmin and factor Xa (fXa). Serp-1 has proven highly potent anti-inflammatory and anti-atherogenic activity. Here we assess the effects of NSP treatment on plaque growth and T-helper (Th) lymphocyte activity in a mouse aortic allograft transplant model, with comparison to Serp-1. NSP and Serp-1 both significantly reduced plaque growth and T-cell invasion. T-bet (a Th1 differentiation marker) was significantly reduced in transplanted aorta with associated reductions in Th1 and Th17, but not Th2, in splenocytes. NSP had additional Th modifying activity in non-transplanted mice. In summary, this is the first report that NSP possesses anti-inflammatory activity in systemic arteries, modifying Th cell responses and significantly reducing plaque growth in mouse aortic allografts.

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BackgroundBinding of chemokines to glycosaminoglycans (GAGs) is classically described as initiating inflammatory cell migration and creating tissue chemokine gradients that direct local leukocyte chemotaxis into damaged or transplanted tissues. While chemokine-receptor binding has been extensively studied during allograft transplantation, effects of glycosaminoglycan (GAG) interactions with chemokines on transplant longevity are less well known. Here we examine the impact of interrupting chemokine-GAG interactions and chemokine-receptor interactions, both locally and systemically, on vascular disease in allografts.Methodology/Principal FindingsAnalysis of GAG or CC chemokine receptor 2 (CCR2) deficiency were coupled with the infusion of viral chemokine modulating proteins (CMPs) in mouse aortic allograft transplants (n = 239 mice). Inflammatory cell invasion and neointimal hyperplasia were significantly reduced in N-deacetylase-N-sulfotransferase-1 (Ndst1 f/f TekCre +) heparan sulfate (GAG)-deficient (Ndst1−/−, p<0.044) and CCR2-deficient (Ccr2−/−, p<0.04) donor transplants. Donor tissue GAG or CCR2 deficiency markedly reduced inflammation and vasculopathy, whereas recipient deficiencies did not. Treatment with three CMPs was also investigated; Poxviral M-T1 blocks CC chemokine receptor binding, M-T7 blocks C, CC, and CXC GAG binding, and herpesviral M3 binds receptor and GAG binding for all classes. M-T7 reduced intimal hyperplasia in wild type (WT) (Ccr2+/+, p≤0.003 and Ccr2−/−, p≤0.027) aortic allografts, but not in Ndst1−/− aortic allografts (p = 0.933). M-T1 and M3 inhibited WT (Ccr2+/+ and Ndst1 +/+, p≤0.006) allograft vasculopathy, but did not block vasculopathy in Ccr2−/− (p = 0.61). M-T7 treatment alone, even without immunosuppressive drugs, also significantly prolonged survival of renal allograft transplants (p≤0.001).Conclusions/SignificanceInterruption of chemokine-GAG interactions, even in the absence of chemokine-receptor blockade, is a highly effective approach to reduction of allograft rejection, reducing vascular inflammation and prolonging allograft survival. Although chemokines direct both local and systemic cell migration, interruption of inherent chemokine responses in the donor tissue unexpectedly had a greater therapeutic impact on allograft vasculopathy.

We report hydrogen bonded capsules with the built-in ability to release loaded bioactive molecules at a physiological temperature of 37 °C.

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