Percutaneous coronary intervention (PCI) or balloon angioplasty alone (POBA = plain old balloon angioplasty) provides relief from angina pectoris in patients with single and multi-vessel coronary artery disease (CAD). Angiographic renarrowing at the site of a previous PCI is known as restenosis and occurs in approximately 30 - 50% of patients at 6 months after the procedure [1 Schwartz RS, Holmes DR Jr, Topol EJ. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms J Am Coll Cardiol 1992; 20(5 ): 1284-93.]. Restenosis after PCI/POBA is the major problem limiting long-term efficacy of this procedure. With the use of stents (a bare metal strut) delivered on an angioplasty balloon, the restenosis rate has been successfully lowered by roughly 10% (20 - 40% restenosis dependent upon the type of lesion and acuity) when compared to balloon angioplasty alone [2 Mitra AK, Agrawal DK. In stent restenosis: bane of the stent era J Clin Pathol 2006; 59(3 ): 232-9.,3 Williams DO, Holubkov R, Yeh W, et al. Percutaneous coronary intervention in the current era compared with 1985-1986: the National Heart, Lung, and Blood Institute Registries Circulation 2000; 102(24 ): 2945-51.]. The advent of drug eluting stents (DES) coated with agents that interfere with cellular proliferation (sirolimus or rapamycin and paclitaxel) has further reduced the restenosis rate to 10-15%, but episodes of acute late stent thrombosis have occurred with inadequate antiplatelet therapy or resistance to platelet inhibition. Currently, the DES has become one of the principal treatments for coronary artery stenosis, together with bare metal stents (BMS) both of which carry some risk of in stent restenosis (ISR) and/or sudden thrombosis [4 Htay T, Liu MW. Drug-eluting stent: a review and update Vasc Health Risk Manag 2005; 1(4 ): 263-76.]. Thus while improved, the problem of restenosis and/or thrombosis persists. Acute thrombotic events are postulated to be caused by a lack of adequate reendothelialization, protective cell covering of the metal stent struts embedded in the artery wall as well as inflammatory reactions to the metal and the DES polymer drug coating. Due to the inherent risk of ISR with the use of stents, POBA is used to alleviate ISR thus preventing two layers of stents. Despite the well-documented track record of reducing ISR using these procedures, it is important to continue investigating other means of treatment.

Although the typical restenotic lesion is different from the atherosclerotic plaque in architecture and composition, both restenotic and atherosclerotic plaques contain smooth muscle cells and fibrous tissue, as well as infiltrating inflammatory macrophages and T cells. After vascular injury is induced by PCI/ POBA, the activation of the inflammatory response is followed by intimal smooth muscle cell proliferation and migration, extracellular matrix formation and neointimal hyperplasia [5 Farb A, Sangiorgi G, Carter AJ, et al. Pathology of acute and chronic coronary stenting in humans Circulation 1999; 99(1 ): 44-52.]. These events are initiated by both cellular and clot forming components of the blood, damaged vascular endothelial cells, connective tissue exposed by balloon denudation, and inflammatory cell mediators [6 Liu MW, Roubin GS, King SB 3rd. Restenosis after coronary angioplasty. Potential biologic determinants and role of intimal hyperplasia Circulation 1989; 79(6 ): 1374-87.]. A critical stage in the development of restenosis is thought to be the activation of an inflammatory response, including platelet adhesion, platelet aggregation, fibrin (clot) deposition, and leukocyte infiltration [7 Hermans WR, Rensing BJ, Strauss BH, Serruys PW. Prevention of restenosis after percutaneous transluminal coronary angioplasty: the search for a "magic bullet" Am Heart J 1991; 122(1 Pt 1 ): 171-87.]. This inflammatory response also involves enzymatic activation of serine protease activity and its subsequent regulation by serine protease inhibitors [8 Altieri DC. Proteases and protease receptors in modulation of leukocyte effector functions J Leukoc Biol 1995; 58(2 ): 120-7.]. One of the serine protease inhibitors, plasminogen inhibitor-1 (PAI-1), has been demonstrated to play a significant role in suppression of intimal hyperplasia in some animal models [9 Carmeliet P, Moons L, Lijnen R, et al. Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice Circulation 1997; 96(9 ): 3180-91.]. PAI-1 knockout mice showed accelerated intimal hyperplasia as observed histologically [10 Carmeliet P. Insights via t-PA and u-PA transgenic mice Restenosis Summit VII 1995; 7: 250-3.,11 Carmeliet P, Schoonjans L, Kieckens L, et al. Physiological consequences of loss of plasminogen activator gene function in mice Nature 1994; 368(6470 ): 419-24.]. Similarly, inhibition of plasminogen with traexamic acid reduced smooth muscle cell migration following arterial balloon injury in rats [12 Jackson CL, Reidy MA. The role of plasminogen activation in smooth muscle cell migration after arterial injury Ann N Y Acad Sci 1992; 667: 141-50.]. Coagulation and fibrinolytic enzymes are known to induce monocyte motility, chemotaxis, tissue infiltration, cell proliferation, and apoptosis [8 Altieri DC. Proteases and protease receptors in modulation of leukocyte effector functions J Leukoc Biol 1995; 58(2 ): 120-7.,10 Carmeliet P. Insights via t-PA and u-PA transgenic mice Restenosis Summit VII 1995; 7: 250-3.,11 Carmeliet P, Schoonjans L, Kieckens L, et al. Physiological consequences of loss of plasminogen activator gene function in mice Nature 1994; 368(6470 ): 419-24.,12 Jackson CL, Reidy MA. The role of plasminogen activation in smooth muscle cell migration after arterial injury Ann N Y Acad Sci 1992; 667: 141-50.]. The search for an agent that prevents restenosis after PCI still represents an important area of biomedical research. Anticoagulants, anti-platelet agents, calcium channel blockers, lipid-lowering agents, steroidal and non-steroidal anti-inflammatory drugs have all been unsuccessful in the prevention of restenosis [7 Hermans WR, Rensing BJ, Strauss BH, Serruys PW. Prevention of restenosis after percutaneous transluminal coronary angioplasty: the search for a "magic bullet" Am Heart J 1991; 122(1 Pt 1 ): 171-87.].

Viruses have complicated lifestyles and have been used to develop many tools used in biological research. Lentiviruses, adenoviruses and retroviruses have all been genetically modified to exploit their gene transfer abilities and prevent neointimal hyperplasia in animal models [13 Cefai D, Simeoni E, Ludunge KM, et al. Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo J Mol Cell Cardiol 2005; 38(2 ): 333-44.,14 Crook MF, Akyurek LM. Gene transfer strategies to inhibit neointima formation Trends Cardiovasc Med 2003; 13(3 ): 102-6.]. Large DNA viruses, especially poxviruses and herpesviruses, have a wide array of strategies to modulate and/or evade host immune and inflammatory responses [13 Cefai D, Simeoni E, Ludunge KM, et al. Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo J Mol Cell Cardiol 2005; 38(2 ): 333-44.,15 Liu L, Lalani A, Dai E, et al. The viral anti-inflammatory chemokine-binding protein M-T7 reduces intimal hyperplasia after vascular injury J Clin Invest 2000; 105(11 ): 1613-21.]. A previous study using attenuated herpes simplex virus 1 mutant R7020, was capable of significantly reducing neointimal hyperplasia in New Zealand White rabbits receiving balloon angioplasty [16 Skelly CL, Chandiwal A, Vosicky JE, Weichselbaum RR, Roizman B. Attenuated herpes simplex virus 1 blocks arterial apoptosis and intimal hyperplasia induced by balloon angioplasty and reduced blood flow Proc Natl Acad Sci USA 2007; 104(30 ): 12474-8.]. Myxoma virus, a poxvirus, encodes a viral anti-inflammatory protein called M-T7 [17 Upton C, Mossman K, McFadden G. Encoding of a homolog of the IFN-gamma receptor by myxoma virus Science 1992; 258(5086 ): 1369-72.]. This protein is capable of mimicking the IFN-γ receptor and binds to a variety of human, rabbit and mouse C, C-C, and CXC chemokines [18 Lalani AS, McFadden G. Secreted poxvirus chemokine binding proteins J Leukoc Biol 1997; 62(5 ): 570-6.]. A study using purified M-T7 protein revealed reduced macrophage invasion due to the inhibition of chemokines in both rats and rabbits receiving vascular injury [15 Liu L, Lalani A, Dai E, et al. The viral anti-inflammatory chemokine-binding protein M-T7 reduces intimal hyperplasia after vascular injury J Clin Invest 2000; 105(11 ): 1613-21.]. The myxoma virus also encodes a secretory serine protease inhibitor (termed Serp-1) that upon infection inhibits serine proteases such as plasminogen [19 Macen JL, Upton C, Nation N, McFadden G. SERP1, a serine proteinase inhibitor encoded by myxoma virus, is a secreted glycoprotein that interferes with inflammation Virology 1993; 195(2 ): 348-63., 20 Upton C, Macen JL, Wishart DS, McFadden G. Myxoma virus and malignant rabbit fibroma virus encode a serpin-like protein important for virus virulence Virology 1990; 179(2 ): 618-31.]. An earlier study [21 Lucas A, Liu L, Macen J, et al. Virus-encoded serine proteinase inhibitor SERP-1 inhibits atherosclerotic plaque development after balloon angioplasty Circulation 1996; 94(11 ): 2890-900.] has demonstrated the effect of the Serp-1 glycoprotein on neointimal hyperplasia after balloon angioplasty in rabbits, showing a significant reduction in neointimal formation and macrophage invasion. A different myxoma virus protein called Serp-2 is a cross-class serpin that is present inside virus-infected cells. In rats, Serp-2 has been shown to significantly reduce plaque size at the site of angioplastic injury [22 Viswanathan K, Bot I, Liu L, et al. Viral cross-class serpin inhibits apoptosis, inflammation and atherosclerosis In: Modulation of Atherothrombotic Factors: Novel Strategies for Plaque Stabilization. The Netherlands: Partners Ipstamp Enschede 2005.].

As an extension to the M-T7, Serp-1 and Serp-2 studies from myxoma virus, we decided to evaluate whether a systemic poxvirus infection could induce sufficient host-immune modulation and/or suppression to elicit antiproliferative effects in a large animal model of restenosis. The porcine model was utilized due to the fact that the neointimal lesion that results after balloon-induced vascular injury is composed of smooth muscle and inflammatory cells, similar to that observed in human patients. Additionally, a species specific poxvirus is available to evaluate these observations. This was a critical issue as most of the known natural immunomodulators, including cytokines, are mainly species specific. This experimental model appears to be very useful in spite of the fact that neither swine cytokines nor swinepox virus (SPV) secretory proteins are well-characterized. In this report we demonstrate that the systemic poxvirus infection induces sufficient host immunosuppression and/or modulation to elicit an antiproliferative effect in a large animal model of neointimal hyperplasia.

Initiation of neointimal proliferation following PCI/ POBA involves activation of inflammatory and thrombotic cascades, which are commonly regulated by serine protease enzymes and inhibitors [8 Altieri DC. Proteases and protease receptors in modulation of leukocyte effector functions J Leukoc Biol 1995; 58(2 ): 120-7.]. Inflammatory cascades result in the adhesion of leukocytes, induced by cytokine-leukocyte signaling. Many poxviruses encode proteins which inhibit or bind to host cytokines. Those proteins are known as immunomodulatory proteins. Myxoma virus produces serine protease inhibitors (Serp-1 and Serp-2) that upon infection inhibits serine proteases such as plasminogen and granzyme B [19 Macen JL, Upton C, Nation N, McFadden G. SERP1, a serine proteinase inhibitor encoded by myxoma virus, is a secreted glycoprotein that interferes with inflammation Virology 1993; 195(2 ): 348-63.,20 Upton C, Macen JL, Wishart DS, McFadden G. Myxoma virus and malignant rabbit fibroma virus encode a serpin-like protein important for virus virulence Virology 1990; 179(2 ): 618-31.]. Local introduction of purified immunomodulatory proteins encoded by vaccinia virus and myxoma virus has been reported to inhibit neointimal hyperplasia induced by vascular injury [21 Lucas A, Liu L, Macen J, et al. Virus-encoded serine proteinase inhibitor SERP-1 inhibits atherosclerotic plaque development after balloon angioplasty Circulation 1996; 94(11 ): 2890-900., 22 Viswanathan K, Bot I, Liu L, et al. Viral cross-class serpin inhibits apoptosis, inflammation and atherosclerosis In: Modulation of Atherothrombotic Factors: Novel Strategies for Plaque Stabilization. The Netherlands: Partners Ipstamp Enschede 2005.]. In our study, based on the idea that all poxviruses secrete some sort of immunomodulatory polypeptides upon infection, an SPV systemic infection was assumed to secrete similar polypeptides. Since these SPV encoded polypeptides have not been characterized, we used systemic SPV infection on domestic feeder pigs to determine if SPV induced immunosuppression could cause an antiproliferative effect in a porcine model of injury-induced neointimal proliferation which mimics restenosis in humans.

In order to determine the degree of neointimal proliferation, at three weeks post-infection with either vehicle or SPV, pig LAD coronary arteries were harvested and morphologically examined. Subcutaneous administration of SPV to domestic feeder pigs a week prior to POBA balloon over-inflation reduced neointimal hyperplasia by 23% compared to mock-infected pigs. When the NI/M ratio was calculated, the magnitude of neointimal proliferation was 22% lower in the SPV-infected group compared to the mock-infected group (Table 1), however this data did not reach statistical significance.

As mentioned previously, 3 out of 8 control pigs developed skin rashes throughout the inguinal region, which resembled swinepox infection. Since swinepox is an endemic disease among swine populations, there is the possibility that those control pigs were cross contaminated or infected before the experiment. Thus, re-analysis of the data in which the 3 control pigs presenting with typical SPV-like infection are included in the SPV group. This generated a further reduction in the NI/M ratio for the SPV-infected pigs compared to control pigs, representing a 33% reduction which reached statistical significance, despite the relatively small population sizes.

The unavailability of reproducible immunological techniques to demonstrate anti-SPV neutralizing antibodies and T cells severely restricted our ability to evaluate experimental SPV infections in pigs. It also restricted our ability to demonstrate whether some pigs were previously immune to SPV infections. Our efforts to establish a reproducible procedure to quantitate anti-SPV neutralizing antibodies were unsuccessful. These observations further confirmed earlier results from some leading SPV laboratories in the U.S. (Richard Moyer and Deoki Tripathy, personal communications). It is not clear if whether SPV itself or the pig immune system contributes to this limitation. However, we were able to confirm active SPV infection based on symptomology and anti-viral antibody detection in serum in all 11 pigs analyzed in subset II. Meanwhile, the 5 control pigs in subset II did not show any evidence of SPV infection using these techniques.

Like the myxoma virus serine protease inhibitor (serpin) Serp-2 that has been shown to reduce inflammatory cell invasion and plaque growth in rodent angioplastic injury models, SPV also encodes a similar serpin called SPV 145R or K1R [22 Viswanathan K, Bot I, Liu L, et al. Viral cross-class serpin inhibits apoptosis, inflammation and atherosclerosis In: Modulation of Atherothrombotic Factors: Novel Strategies for Plaque Stabilization. The Netherlands: Partners Ipstamp Enschede 2005., 25 Afonso CL, Tulman ER, Lu Z, et al. The genome of swinepox virus J Virol 2002; 76(2 ): 783-90., 26 Massung RF, Jayarama V, Moyer RW. DNA sequence analysis of conserved and unique regions of swinepox virus: identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue Virology 1993; 197(2 ): 511-28.]. The SPV145R has a high level of homology with the cowpox virus serpin/crmA homologue. The crmA protein from cowpox virus has been shown to inhibit the cellular IL-1β converting enzyme, modulating inflammation and apoptosis [27 Ray CA, Black RA, Kronheim SR, et al. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin- 1 beta converting enzyme Cell 1992; 69(4 ): 597-604.]. On a different note, the SPV 008 encodes a protein that shares 25% sequence homology the myxoma virus MT-7 protein [25 Afonso CL, Tulman ER, Lu Z, et al. The genome of swinepox virus J Virol 2002; 76(2 ): 783-90.]. The myxoma virus MT-7 protein is an IFN-γ viroceptor and multiple chemokine binding protein, that has been shown to display anti-inflammatory properties in rat and rabbit vascular injury models [15 Liu L, Lalani A, Dai E, et al. The viral anti-inflammatory chemokine-binding protein M-T7 reduces intimal hyperplasia after vascular injury J Clin Invest 2000; 105(11 ): 1613-21., 17 Upton C, Mossman K, McFadden G. Encoding of a homolog of the IFN-gamma receptor by myxoma virus Science 1992; 258(5086 ): 1369-72.]. In addition to the proteins mentioned here, SPV encodes many more immunomodulating proteins that could possibly control inflammation during a systemic infection and therefore reduce neointimal hyperplasia.

We have shown that a systemic infection of swinepox is capable of alleviating neointimal hyperplasia induced by angioplastic vascular injury. Although the actual proteins and mechanisms responsible for these observations are unknown, it suggests that a systemic infection of a poxvirus is capable of generating therapeutic effects for its host. Additional experiments knocking out selected SPV genes potentially involved in immunomodulation will precisely identify proteins which participate, either directly or indirectly, in the suppression of neointimal hyperplasia in pigs.