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Aim: Ser ine p rotease in hibitors ( Serpins ) regulate key clotting and inflammatory pathways. Two anti-inflammatory Serpins , neuroserpin (NSP) and Serp-1 markedly reduce arterial inflammation and plaque growth in a rodent aortic transplant vasculitis model after single dose infusion. NSP is a mammalian serpin that inhibits thrombolytic proteases and Serp-1 is a virus-derived serpin that inhibits both thrombotic and thrombolytic proteases. We postulated that proteolytic cleavage of the reactive site loop (RSL) produces active peptide derivatives with expanded functions. 8 peptide sequences encompassing predicted cleavage sites for Serp-1 and NSP were synthesized and tested in mouse aortic allograft transplant and herpesvirus (MHV68) vasculitis models. The main aim was to assess therapeutic potential for RSL peptides derived from NSP and Serp-1 in vascular inflammation. Methods: We assessed the capacity of 8 predicted serpin RSL peptides, 4 from Serp-1 and 4 from NSP to reduce vascular inflammation. 40 C57BL/6 mice had aortic allograft transplant with a single IV injection of each single peptide. 29 IFN-γR KO mice had MHV-68 infection and were treated for 10 days with S-2 (ISRMAV), S-7 (GTTASSDTAITLIPR), Inverse S-7 (RPILTIATDSSATTG), S-8 (MAVLYPQVIVDHPFFFLIRNR) peptides, or Saline control. Results: RSL peptides with R or RN sequences, but not RM, and with 0-+1 charge significantly reduced vascular inflammatory cell invasion and plaque growth after aortic transplant from C57Bl/6 donor to BALB/c recipient transplant mice (5-6 transplants/peptide) (P < 0.01). S-1, S-3, S-7 & S-8 peptides reduced inflammation and plaque in aortic transplant. Hydrophobicity and negative charge in S-6 peptide increased thrombosis. S-7 peptide also significantly improved survival in a lethal MHV68 infection vasculitic model (150 days survival; P < 0.043), while inactive S-2 peptide, Inverse S-7 and S-8 had no effect (P = NS; 40 days) (P< 0.043). Conclusions: In conclusion, a series of serpin RSL peptides were synthesized based upon natural protease cleavage sites. RSL peptides have demonstrated significant efficacy in reducing inflammatory vasculitis after transplant or after lethal mouse herpesviral (MHV) infections, extending serpin regulatory activity.

Introduction: Periodontal disease (PD) is a polymicrobial dysbiotic chronic inflammatory disease produced by a complex subgingival microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). American Heart Association supported the association between PD and ASVD but not causal association. Prior reports investigated the associations between these two diseases PD and ASVD. But the complex signaling molecules and mechanisms establishing a causal association are not well defined. Methods: To investigate how polybacterial infections initiate atherosclerotic plaque growth, ApoE null mice were orally infected with a polybacterial consortium of 4 well-characterized periodontal pathogens Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum . We assessed PD characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Results: Polybacterial infections have enhanced gingival colonization and significantly induced alveolar bone loss ApoE null mice. Polybacterial infections were found to be invasive with hematogenous dissemination into heart and aorta. Polybacterial infection-induced significantly higher levels of serum risk factors (oxidized LDL, nitric oxide), altered lipid profiles (cholesterol, triglycerides, chylomicrons, VLDL) as well as accelerated aortic plaque formation in ApoE null mice. Polybacterial infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Gingival infections after 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. At 24 weeks of chronic infection additional changes in inflammatory molecules were induced with reduced KC, MCSF, and enhanced GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, and RANTES. Conclusions: This is the first study demonstrating differences in the host immune response to a polybacterial infection with atherosclerotic lesion progression in a mouse model.

Background: Transplant allograft vasculopathy (TAV) is the leading cause for late transplant organ loss. TAV is induced by activation and invasion of inflammatory monocytes and T cells in response to chemokine binding to glycosaminolycans (GAGs) in the endothelial glycocalyx. We investigated the effect of inhibiting heparin sulfate (HS-GAG) / chemokine interactions on rejection in a renal transplant model with (a) heparin sulfate HS-GAG deficient (Ndst1 -/- ) donor mice and (b) a virus-derived chemokine modulating protein M-T7, that is blocks C, CC and CXC interaction with GAGs. Methods: BALB/c renal allograft recipients receiving either Ndst1 -/- (C57BL/6 background) or C57Bl/6 wild type (WT) donor kidneys (n = 48 each) were treated for 10 days with either Saline, M-T7 or one of three M-T7 point mutations (100 ng/gm) with differing inhibitory actions for HS-GAG / chemokine interaction. No other immunosuppressants were added. Results: Ndst1 -/- kidneys had 10X reduced HS content and markedly reduced sulfation compared to WT kidneys. Overall acute rejection and vasculitis scores were significantly reduced in Ndst1 -/- donor kidneys (P < 0.0001). In WT donors, M-T7 significantly reduced rejection and vasculitis (P < 0.0001), but interestingly, lost activity in Ndst1 -/- allografts. CD3+ cells were significantly reduced (P < 0.0001) in Ndst1 -/- donor grafts and with M-T7 treatment in WT transplants and TH17+ cells were significantly reduced in spleen cell isolates. M-T7 point mutation E 209 I retained activity in both Ndst1 -/- and WT donors while but R 171 E was active only in WT. The third mutant F 137 D was inactive. PCR array analysis detected significant decreases in IL-4, NOS2 and CC chemokine 20, in Ndst1 -/- donor transplants or in WT transplants with M-T7 or M-T7 mutant treatments with significant reductions in TAV. Conclusions: A critical role played by HS-GAG / chemokine interactions in the DONOR allograft transplant is demonstrated by these studies. Significant decreases in acute rejection and inflammatory vasculitis are seen in Ndst1 -/- donors with saline treatment and after treatment of C57Bl/6 WT donor grafts with M-T7. HS-GAG composition of donor organs has a potential decisive effect on transplant viability.

Background Glucocorticoids are a principal treatment for giant cell arteritis (GCA), with some studies reporting predominate effect on T helper17 (Th17) cell activity in GCA, with lesser effects on other inflammatory cells. Serp-1 is a 55kDa myxomaviral serpin that modifies macrophage, Th1, and Th17 responses in aortic transplants in animal models. Objectives We have analyzed the effects of a virus-derived anti-inflammatory ser ine p rotease in hibitor ( serpin ) on human temporal artery (TA) biopsies isolated from patients with suspected GCA. Methods Using a newly developed “Aortic Window Xenopatch” model, human TA biopsy sections were divided implanted as full thickness grafts into the abdominal aorta of SCID mice in parallel and tested for response to Serp-1, with and without human PBMC infusion (N = 32). Results TA sections positive for arteritis (GCA pos ) displayed significantly increased inflammatory plaque when compared to negative sections (GCA neg ). Serp-1 reduced plaque in GCA pos sections (+ cells after PBMC infusion with reduced TNF-α expression in spleen, but not CD3 + , CCR6 + , nor CD86 + . Splenocytes from mice with GCA pos grafts had increased interleukin-1beta (Il-1β), IL-17, and CD25 expression, while IL-1β was significantly reduced by Serp-1.Serp-1 also markedly reducedt gene expression for the FII, FXa-PAR2, tPA, uPA in splenocytes (P Conclusions Treatment with a virus-derived serpin, significantly reduces inflammation and plaque thickness in human GCA pos TA xenograft implants with associated reductions in macrophage and Th1 and Th17 responses in mice.

Background Lethal viral infections produce widespread inflammation with vascular leaking, clotting and bleeding (DIC), and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by a cytokine storm and also induce systemic inflammation. Normal host ser ine p roteases in hibitor ( serpin ) regulation is lost. Serp-1 is a potent myxomavirus anti-inflammatory protein, which inhibits factor X (fX), tPA and uPA in vitro . Serp-1 reduces arterial inflammation and atherogenesis in many animal models and has been successfully tested in patients with unstable plaque and stent implants. Methods and results Purified Serp-1 protein treatment significantly improved survival in lethal murine Gammaherpesvirus-68 (MHV68) infection in interferon gamma receptor (IFNγR) knock-out mice (N = 72 mice; P < 0.02) when given for 10 up to 30 days after infection. Treatment with a mammalian serpin, neuroserpin, that inhibits only tPA and uPA, was ineffective (N = 30 mice; P = NS). Serp-1 reduced viral antigen, lung hemorrhage, and aortic, lung and colon inflammation in MHV68 infected mice. Neuroserpin suppressed a wide swath of immune cell responses, while Serp-1 selectively increased CD11c+ splenocytes (macrophage and dendritic cells) and reduced CD11b+ tissue macrophage. Serp-1 significantly increased the IL-10 gene expression in aorta and plasma protein, whereas neuroserpin decreased IL-10. Serp-1 mediated survival in MHV68 infected mice was lost after treatment with IL-10 receptor antibody immediately post infection (N = 12 mice; P =0.014). Conclusions Serp-1, a unique myxomavirus-deried serpin, improves survival after lethal mouse herpesviral infection, reduces viral load, hemorrhage and arterial inflammation. Serp-1 provides a potential new therapeutic approach for lethal viral sepsis and DIC through altered FX and IL-10.

Modification of the tumor microenvironment by inflammatory cells represents a newly recognized driving force in cancer with a critical role in tumor invasion, growth, angiogenesis, and metastasis. Increased serine proteases in the thrombolytic cascade, specifically urokinase-type plasminogen activator and its receptor, correlate with inflammatory cell invasion, angiogenesis, cancer growth and invasion. Inflammation in pancreatic cancer is linked with myeloid-derived suppressor cell activity and cancer progression. Myxomavirus is a complex DNA virus encoding highly potent immune modulators such as Serp-1, which inhibits uPA, tPA, plasmin and coagulation factor X. Serp-1 has also been demonstrated to inhibit angiogenesis in a chicken chorioallantoic membrane (CAM) model. To explore the potential use of anti-inflammatory, anti-angiogenesis proteins that target thrombotic and thrombolytic as well as inflammatory pathways, for cancer treatment, severe combined immunodeficient (SCID) mice engrafted with human cancer cells were treated with Serp-1 and neuroserpin, a selective mammalian inhibitor of thrombolytic proteases or control saline. Proteins were tested for efficacy in preventing human pancreatic and breast cancer cell growth in NOD/SCID mice (n=67 total) at 4 weeks after implantation. Serp-1 and neuroserpin inhibited pancreatic cancer Hs766t (n=27; P=0.03 and P=0.01, respectively), but not breast cancer MDA231 growth (n=11) when compared to saline controls (N =5). Serpin inhibition of pancreatic tumor growth was associated with decreased MDSC as shown by flow cytometry analysis of splenocyte (P=0.009). Serpin-mediated anti-inflammatory activity in pancreatic tumor growth was also demonstrated by reduced macrophage infiltration in tumors (P=0.001). Furthermore, RT-PCR array analysis revealed a five-fold decrease in the expression of inducible nitric oxide synthase (iNOS), which has been shown to play important role in tumor angiogenesis. Conclusions Anti-inflammatory protease inhibitors, virus-derived Serp-1 and mammalian neuroserpin, may provide new treatment approaches by targeting the angiogenesis and inflammatory responses that support cancer cell growth.

Vasculitic syndromes cause devastating arterial occlusion with associated end organ ischemia and significant morbidity. Treatment for vasculitic syndromes remains very limited. Gamma 68 herpesvirus (g68HV) infection in interferon gamma receptor deficient (IFNγR -/- ) mice provides a good working model of artery inflammation, inducing early death with associated inflammatory cell invasion. Myxomavirus serpin, Serp-1, inhibits tPA, uPA, plasmin (thrombolytic) and fXa (thrombotic) proteases, reducing inflammation in a wide range of animal models whereas mammalian neuroserpin (NSP) inhibits tPA and uPA and reduced myocardial damage in clinical trial of unstable angina and stent implant. To assess the capacity of serpins to block inflammatory vasculopathy development, γ68HV infected IFNγR -/- mice were treated with either Serp-1 (100ng/g/100μl), NSP (100ng/g/100μl) or saline (100ul) .Serp-1 treatment given by daily intraperitoneal injections (i.p.) for the first 10 days post infection (6 - 12.5 X 106 PFU), significantly prolonged survival in mice (IFNγR -/- ) (P ≤ 0.048). Saline treated γ68HV infected mice survived up to 42 days, whereas Serp-1 treated mice survived up to 55 days (N=60 mice, 6-12 mice per group). NSP treatment (100ng/gm i.p. X 10 days) produced an opposite trend toward increased mortality (P ≤ 0.079; N=16). In a subsequent study, prolonged daily treatment with Serp-1 (100ng/gm i.p.) given for the first 30 days post infection, markedly prolonged survival in γ68HV infected INFγR -/- mice for up to 150 days (P ≤ 0.032, N =10). Treatment with Serp-1, that was started 3 weeks post γ68HV infection, produced a non-significant trend toward improved survival (P=0.685; N = 12 mice), whereas treatment begun at 1 week post infection again significantly improved survival (P ≤ 0.020; N=12 mice). Serp-1 treatment significantly reduced inflammatory cell invasion in the aortic adventitia ( p < 0.010) and lung ( p < 0.047), NSP significantly increased inflammatory cell invasion when compared to Serp-1 treatment in infected mice in the lung interstitial layer (p ≤ 0.005). At 10 days follow up the plasma cytokine interleukin-1beta (IL-1β) was significantly reduced by Serp-1 treatment (p ≤ 0.049), Treatment with the NSP reduced plasma cytokine TNFα ( p ≤ 0.022), IFNγ (p ≤ 0.022), and IL-4 (p ≤ 0.022) in γ68HV infected IFNγR -/- mice (N=14).Gene expression was significantly increased in Serp-1 treated mice for regulatory serpins in the thrombolytic cascade, specifically PAI-1 (p ≤ 0.020) and unexpectedly NSP (p ≤ 0.032), gene expression was significantly reduced for E selectin (p ≤ 0.048) and increased for IL-10 (p ≤ 0.031), and addressin (p ≤ 0.002) with Serp-1 treatment at 10 days . Compared to the saline treated mice, NSP treatment lead to decreased arterial IL-17 and IL-10 gene expression (p ≤ 0.006 and 0.041, respectively) and increased PAI-2 (p ≤ 0.021) . When comparing differences in gene expression for Serp-1 and NSP; Serp-1 treatment increased gene expression for IL-10 (p ≤ 0.001), bFGF (p < 0.022), Addressin (p < 0.001), PAI-1 (p < 0.021), and NSP (p < 0.009) and NSP reduced IL-6 (p < 0.016) and PAI-2 (p < 0.016). Conclusion: Serp-1 but not NSP produced an unexpected improved survival after γ68HV infection with associated rebalancing of the coagulation and thrombolytic cascades as well as modulating inflammatory responses. Anti-inflammatory serpin provides a potential new therapeutic approach to virus induced vasculitis.