Human cytomegalovirus infection and atherothrombosis | SpringerLink

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Dec 13, 2011 - Life-long persistent infection with human cytomegalovirus (HCMV) has been associated with atherosclerosis. In vivo studies have revealed that ...
J Thromb Thrombolysis (2012) 33:160–172 DOI 10.1007/s11239-011-0662-x

Human cytomegalovirus infection and atherothrombosis Milan Popovic´ • Katarina Smiljanic´ • Branislava Dobutovic´ • Tatiana Syrovets Thomas Simmet • Esma R. Isenovic´



Published online: 13 December 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Vascular endothelium, as a key regulator of hemostasis, mediates vascular dilatation, prevents platelet adhesion, and inhibits thrombin generation. Endothelial dysfunction caused by acute or chronic inflammation, such as in atherosclerosis, creates a proinflammatory environment which supports leukocyte transmigration toward inflammatory sites, and at the same time promotes coagulation, thrombin generation, and fibrin deposition in an attempt to close the wound. Life-long persistent infection with human cytomegalovirus (HCMV) has been associated with atherosclerosis. In vivo studies have revealed that HCMV infection of the vessel wall affects various cells including monocytes/macrophages, smooth muscle cells (SMCs) and endothelial cells (ECs). HCMV-infected SMCs within vascular lesions display enhanced proliferation and impaired apoptosis, which contribute to intimamedia thickening, plaque formation and restenosis. Monocytes play a central role in the process of viral dissemination, whereas ECs may represent a viral reservoir, maintaining persistent infection in HCMV-infected atherosclerotic patients following the primary infection. Persistent infection leads to dysfunction of ECs and activates proinflammatory signaling involving nuclear factor jB, specificity protein 1, and phosphatidylinositol 3-kinase, as well as expression of platelet-derived growth factor

M. Popovic´ (&)  K. Smiljanic´  B. Dobutovic´  E. R. Isenovic´ Department for Radiobiology and Molecular Genetics, Vincˇa Institute, University of Belgrade, P.O. Box 522, 11001 Belgrade, Serbia e-mail: [email protected] T. Syrovets  T. Simmet Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm University, 89081 Ulm, Germany

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receptor. Activation of these pathways promotes enhanced proliferation and migration of monocytes and SMCs into the intima of the vascular wall as well as lipid accumulation and expansion of the atherosclerotic lesion. Moreover, HCMV infection induces enhanced expression of endothelial adhesion molecules and modifies the proteolytic balance in monocytes and macrophages. As a consequence, infected endothelium recruits naive monocytes from the blood stream, and the concomitant interaction between infected ECs and monocytes enables virus transfer to migrating monocytes. Endothelial damage promotes thrombin generation linking inflammation and coagulation. HCMV, in turn, enhances the thrombin generation. The virus carries on its surface the molecular machinery necessary to initiate thrombin generation, and in addition, may interact with the prothrombinase protein complex thereby facilitating thrombin generation. Thus, infection of endothelium may significantly increase the production of thrombin. This might not only contribute to thrombosis in patients with atherosclerosis, but might also induce thrombin-dependent proinflammatory cell activation. This review summarizes the existing evidence on the role of HCMV in vascular inflammation. Keywords HCMV  Coagulation  Vascular inflammation  Atherosclerosis Abbreviations Ab Antibody Akt Serine/threonine protein kinase aPL Antiphospholipid ECs Endothelial cells HCMV Human cytomegalovirus EGFR Epidermal growth factor receptor HSV-1 Herpes simplex virus 1 (human herpes virus 1)

HCMV in atherothrombosis

HSV-2 IL-1a ICAM-1 MAPK M-CSF NF-jB PI3K PDGFR PDGFR-b proPL Sp1 SMCs VCAM-1 VSMCs

Herpes simplex virus 2 (human herpes virus 2) Interleukin-1a Intercellular adhesion molecule-1 Mitogen activated protein kinase Macrophage colony-stimulating factor Nuclear factor-jB Phosphatidylinositol 3-kinase Platelet-derived growth factor receptor Platelet-derived growth factor receptor-b Procoagulant phospholipid Specificity protein 1 Smooth muscle cells Vascular cell adhesion molecule-1 Vascular smooth muscle cells

Introduction Endothelium plays a pivotal role in the maintenance and modulation of vascular homeostasis both under physiological as well as under pathophysiological conditions [1]. Under physiological conditions, endothelium inhibits coagulation and promotes vascular dilatation [2, 3] thereby preserving an anti-inflammatory state [4, 5]. The antiinflammatory endothelium efficiently scavenges free radicals [6–8], it prevents thrombin generation [9], inhibits platelet activation and adhesion [10]. Similarly, it reduces adhesion and transmigration of inflammatory leukocytes [11, 12]. By contrast, under pathological conditions, which prevail in patients with advanced atherosclerosis, diabetes, or chronic arterial hypertension [2, 13, 14], endothelium acquires a proinflammatory phenotype. This state is characterized by increased expression of adhesion molecules, chemokines, cytokines, and growth factors, which promote homing of circulation leukocytes to the sites of inflammation [15, 16]. Human cytomegaloviruses (HCMV) belong to the b-herpes virus subfamily, which after primary infection persist for the life span of the host [17]. In neonates primary HCMV infection may cause life-threatening diseases and it may be associated with several neurological birth defects [17]. Whereas in healthy, immunocompetent individuals, HCMV infection remains mainly asymptomatic, persistent or recurrent infection of immunocompromised individuals such as acquired immune deficiency syndrome patients [18, 19] or transplant organ recipients may result in significant morbidity and mortality [20–23]. Chronic infection of the vasculature with HCMV has been linked to the development of cardiovascular diseases such as atherosclerosis, restenosis, and transplant vascular sclerosis [24–27].

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Cellular effects of HCMV Endothelial cells (ECs) represent a primary site of HCMV infection during primary infection [28–30] and serve as a possible viral reservoir essential for viral spread and persistence [30]. Apart from ECs, HCMV exploits epithelial cells, smooth muscle cells (SMCs), and fibroblasts as additional target cells for viral replication [20, 30–32]. At neutral pH, HCMV enters cells via direct fusion of the viral envelope with the plasma membrane [33]. However, in some cell lines such as retinal pigment epithelial cells and ECs, virus entry is accomplished via receptormediated endocytosis taking place at low pH [34]. Indeed, HCMV entry is a highly complex process requiring multiple envelope glycoproteins and a series of host receptors. Of all glycoproteins encoded by the virus, five appear to be essential for the virus replication in vitro [35]; these include gB (UL55) [35], gM/gN (UL100/UL73) [36, 37], and gH/gL (UL75/UL115) [38]. Infection of ECs triggers induction of proinflammatory adhesion molecules such as vascular adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and the activation marker CD40 [39, 40]. Consistently, activation of ECs promotes adhesion of leukocytes and platelets [39, 41, 42]. Binding of HCMV to ECs also induces an angiogenic response via PI3K and the mitogen activated protein kinase (MAPK) signaling pathway [43]. This angiogenic response to infection is considered to contribute to HCMV-associated vascular disease. Indeed, impaired regulation of angiogenesis upon viral infection may lead to development of atherosclerotic plaques within the vascular wall [43]. In fibroblasts, neutralizing antibodies against EGFR inhibited virus attachment and entry suggesting that EGFR may act as a HCMV entry receptor in these cells [44]. More recently, other studies devoted to the role of EGFR in HCMV entry and signaling in fibroblasts demonstrated that HCMV did not induce activation of EGFR [45, 46]. In line with this observation, an EGFR kinase inhibitor was unable to inhibit the HCMV-induced gene expression in fibroblasts [46]. Additionally, the same EGFR neutralizing monoclonal antibody that had previously been reported to inhibit HCMV attachment and entry [44], did not reduce virus entry in pretreated fibroblasts [46]. Further, when ECs and epithelial cells were pretreated with the EGFR-neutralizing antibodies and then challenged with clinical isolates of HCMV, no decrease in virus gene expression could be detected [46]. Thus, considering the disparity between these studies [44–46], as well as the ability of HCMV to downregulate EGFR expression more work is needed to definitively clarify the putative contribution of EGFR to HCMV entry. HCMV infection was shown to initiate activation of epidermal growth factor receptor (EGFR) and the robustly

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activated EGFR kinase stimulates downstream signaling, involving the serine/threonine protein kinase (Akt), phosphatidylinositol 3-kinase (PI3K), phospholipase C-c, as well as mobilization of intracellular Ca2? [44]. Consistently, a specific EGFR kinase inhibitor blocked the HCMV-induced gene expression in an EGFR-expressing cell line [44]. In infected humans, ECs are reported to be a natural site of HCMV infection during primary infection [8, 30] and a possible viral reservoir essential for viral spread and persistence [29]. In addition to ECs, HCMV exploits epithelial cells, SMC, and fibroblasts as important target cells for virus replication [8]. Infection of ECs is reported to lead to the activation of proinflammatory adhesion molecules such are VCAM-1 and ICAM-1 and activation marker CD40 [32, 38]. Accordingly, activation of ECs results in potentiated adhesion of leukocytes and platelets [39, 41]. Wang et al. showed that virus infection initiates activation of EGFR [44]. The authors demonstrated that robustly activated EGFR kinase triggers downstream signaling pathway, involving a Akt, PI3K, phospholipase C-c, as well as mobilization of intracellular Ca2? [44]. Additionally, use of specific EGFR kinase inhibitor blocked HCMV gene expression in EGFR-expressing cell line [44]. Moreover, in fibroblasts, neutralizing antibodies against EGFR, inhibited virus attachment and entry, suggesting that EGFR may act as HCMV entry receptor in fibroblasts [44]. More recently, two other studies have been conducted investigating the role of EGFR in HCMV entry and signaling [29, 30]. It was demonstrated that HCMV did not induce activation of EGFR and an EGFR kinase inhibitor was not able to inhibit HCMV gene expression in fibroblast cells [29, 30]. Additionally, the same EGFR neutralizing monoclonal antibody that had previously been reported to inhibit HCMV attachment and entry [44] did not reduce virus entry in pretreated fibroblasts [46]. Further in the same study when epithelial cells and ECs were pretreated with EGFR-neutralizing antibodies and then challenged with a clinical isolates of HCMV, no decrease in virus gene expression was detected [46]. Thus, considering, the disparity between these studies [44–46], and ability of HCMV to downregulate of EGFR expression more work needs to be done to discover the contribution of EGFR to HCMV entry. In vascular SMCs (VSMCs), HCMV infection could interfere with vascular functions by affecting the expression of platelet-derived growth factor receptor (PDGFR) [47, 48]. The normal propensity of SMCs to migrate toward the neointima of injured vessels is enhanced by HCMV infection, in part as a result of increased PDGFR expression [47]. The increased PDGF signaling caused by increased PDGFR-b expression could also account for an augmented proliferation of SMCs. The enhanced SMC migration and increased proliferation would contribute to

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both, increased neointima development and lesion expansion. Although initial data had been obtained in rodent models, it has recently been confirmed that HCMV infection of SMCs from human coronary arteries also leads to enhanced expression of PDGFR-b [48]. In addition, HCMV infection alters cellular metabolism, promoting accumulation of cholesterol and cholesteryl esters [49] and it increases expression and activity of scavenger receptor in VSMCs [50], events that may favor the development of vascular lesions. In monocytes, HMCV infection provokes a massive inflammatory response involving nuclear factor jB (NF-jB), specificity protein 1 (Sp1), and PI3K signaling pathways [51]. HCMV induces overexpression of adhesion molecules in ECs and modifies the proteolytic balance in monocytes [52] and in macrophages [53]. Infected ECs may then recruit naive monocytes, which become not only activated, but also infected whereas they migrate through EC junctions into the surrounding tissue, at the same time spreading the virus from blood and ECs into the surrounding tissue [52]. Transfer of the virus is increased due to increased expression of adhesion molecules by the infected ECs, by the increased permeability, the decreased actin stress fiber formation, and the altered expression of tight and adherence junction proteins. The consequence of these processes is an effective hematogenous virus spreading. This mechanism is also expected to promote the vascular diseases associated with HCMV infection [52]. Propagation of HCMV in cell culture has been an important research tool to dissect the virus-triggered intracellular events in infected cells. Of course, studies on HCMV in cell culture have limitations, in part because propagation under laboratory conditions may foster selection of viruses adapted for growth under in vitro conditions [54]. Indeed, commonly used laboratory strains have multiple deletions, rearrangements [55, 56], and other mutations stressing the importance of clinical observations for validation of these results. HCMV in thrombosis Whether or not infectious agents are involved in the pathogenesis of atherothrombosis has been a matter of intensive debate for the past two decades. Although there is no definite proof of a causal role of HCMV in atherothrombosis, members of the herpes virus family are known to rapidly convert vascular ECs from a noncoagulant to a procoagulant phenotype [42, 57–61]. This inflammatory effect has received considerable attention because thrombin generated in such an environment is capable of inducing adherence and recruitment of inflammatory cells resulting in tissue damage [62]. Hence, thrombin has been proposed as a possible causative agent in the initiation and/or promotion

HCMV in atherothrombosis

of atherogenesis [42, 63, 64]. The involvement of thrombin in herpes virus pathology has clinically been implicated by fibrin deposits in the microvasculature of mucosal lesions [65], further by the development of disseminated intravascular coagulation [66], and by the observation of accelerated atherosclerosis following organ transplantation in HCMV-infected patients [67–69]. Indeed, a cause—and— effect relationship has been established in avians, in Marek’s disease herpes-type virus infections [70, 71], and in rat models of CMV infection [72], where thrombosis and atherosclerosis significantly correlate with the infection. The question whether or not HCMV infection directly affects thrombin generation, still remains unanswered. Some data suggest that purified HCMV can directly interact with prothrombinase proteins and substitute for synthetic procoagulant phospholipid (proPL) vesicles to catalyze the generation of thrombin [73]. Furthermore, dense bodies and noninfectious enveloped particles, which coegress with virions from host cells, express similar procoagulant activity [74, 75]. It was also shown that CMV infection of rat aortic ECs cause the appearance of procoagulant activity on these cells later, and in line with this observation, it was demonstrated that herpes virus infection of ECs promoted enhanced monocyte-endothelial adhesion [63, 76]. Interestingly, this enhanced adhesion was blocked by monoclonal antibodies directed against the viral-encoded cell surface glycoprotein gC. In addition, the adhesion was also blocked by treatment of ECs with specific thrombin inhibitors or by growing cells in prothrombindepleted serum [63, 76]. These experiments also revealed that bound gC promotes activation of factor X on the infected ECs, thereby contributing to thrombin generation [63, 76]. Interestingly, HCMV as well as two other members of the herpes virus family, namely HSV-1 and HSV-2, possess the molecular machinery for the initiation of thrombin generation on their envelope surface [60]. The activation complex apparently includes endogenous proPL, e.g., phosphatidylserine, to facilitate the enzyme–cofactor– substrate complex assembly, as well as tissue factor, which participates in factor X activation [60]. Because neither the HCMV nor HSV genome encode a tissue factor homologue, it must be acquired along with proPL from a host cell during formation of the virus envelope [60]. Additional components present on the virus surface may also contribute to thrombin production [60, 61]. The HSV-1encoded glycoprotein C is also expressed on the surface of infected ECs, and has been shown to accelerate factor X activation [60]. Whether it can act in a cell-bound form within tenase, has not yet been determined, but this possibility would provide an explanation for the incomplete inhibition of factor Xa generation observed in the presence of anti-tissue factor monoclonal antibodies. The fact that direct generation of factor Xa by virus alone was not

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observed, and that the thrombin generation was not inhibited by a tissue factor antibody, suggests that factor FVIIa might be involved [60] (Fig. 1). In addition, our recent in vitro study demonstrates that thrombin might trigger profound proinflammatory activation of ECs pre-exposed to HCMV [77]. Thus, in human umbilical vein ECs pretreated with HCMV, thrombin induces PAR3-mediated expression of proinflammatory IL-1a [77] and M-CSF mRNA [77]. Our results combined with the existing literature [60, 61] led us to conclude that endothelial infection might significantly contribute to the proinflammatory status of the endothelium, most likely by enhancing thrombin generation, thereby leading to endothelial dysfunction [77]. HCMV infection of ECs may lead to vascular damage and coagulation in vivo. A case relating HCMV infection to thrombosis was first reported in 1997 when in a 31-yearold women development of acute portal vein thrombosis was observed in the course of acute HCMV infection [78]. Others have shown that the viral infection of ECs activates the coagulation cascade and contributes to thrombus formation and acute vascular events in patients with atherosclerotic disease [42]. The increased thrombogenicity was dependent on active virus replication and could be inhibited by the antiviral drugs foscarnet and ganciclovir. These results indicate that a late viral gene may mediate this phenomenon [42]. Currently, there are only a few reports on concomitant acute HCMV infection and thrombosis in immunocompetent patients [79]. However, a recent case report on a previously healthy adult without any history of vasculitis or thromboembolic disease, describes HCMV infection complicated by extensive mesenteric arterial and venous thrombosis [80]. This was the first description of thrombosis in a HCMV-infected immunocompetent individual who had no predisposing risk factors for thrombosis. Another two case reports described acute HCMV infections in non-immunocompromised adults that were complicated by venous thrombosis with pulmonary embolism [81]. There were also several reports on acute HCMV infection and splenic infarcts in immunocompetent patients [82]. In addition, in an immunocompetent female patient with acute HCMV infection an association with concomitant thrombosis was reported, a syndrome that had previously only been recognized in immunocompromised patients [83]. Similarly, the case of a previously healthy adult with cytomegalovirus infection was complicated by tibiopopliteal deep venous thrombosis and associated with a Factor V Leiden heterozygous mutation [84]. This case report emphasizes the involvement of HCMV in induction of vascular thrombosis in patients with predisposing risk factors for thrombosis [84]. In another case report, pulmonary embolism was associated with HCMV infection and the presence of

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COAGULATION VASCULAR INFLAMMATION

INCREASED INFLAMMATORY RESPONSE HCMV DISSEMINATION

HCMV INFECTION

+ NF-κB

FIIa

+ Sp1

+ PI3K FII

+ Akt

FVIIa Naive Mo

FXa

+ IL-1α, PDGFR, ICAM-1, CD40L, VCAM-1

FX +

+ M-CSF, EGFR Mo ADHESION

Endothelial cell

Monocyte

Human cytomegalovirus

Fig. 1 Proposed mechanisms for CMV-induced vascular inflammation. Square with plus symbol indicates induction; upward arrows indicate elevated levels; downward arrows indicate adhesion initiation; Mo monocyte, IL interleukin, M-CSF macrophage colonystimulating factor, NF-jB nuclear factor-jB, PI3K phosphatidylinositol 3-kinase, Akt serine/threonine protein kinase, Sp1 specificity

protein 1, EGFR epidermal growth factor receptor, PDGFR plateletderived growth factor receptor, ICAM-1 intercellular adhesion molecule-1, VCAM-1 vascular cell adhesion molecule-1, CD40L CD40 ligand, FX, VII, II factor X, VII, II, Xa, FXa, VIIa, IIa activated factor X, VII, II, CMV human cytomegalovirus

antiphospholipid (aPL) antibodies in an immunocompetent individual [85]. These data support the hypothesis that aPL antibodies may be induced by HCMV infection, and may play a role in the formation of vascular thrombi. It is also plausible that some aPL antibodies induced by viral products are pathogenic in specific groups of predisposed individuals [86]. Yet, further studies are needed to identify the factors of susceptibility to aPL antibodies to finally facilitate preventive treatment. Analysis of the HCMV-associated thrombosis in clinical studies has been reported only sporadically. According to the findings from Atzmony et al., the incidence of thrombosis in a study enrolling 140 patients with acute HCMV infection and 140 consecutive matched patients in whom acute infection had been excluded (control group) was 6.4% [83]. Moreover, acute HCMV infection in this study population was associated with thrombosis independent of other risk factors for thrombosis [83]. Thus, already published data support the notion that HCMV infection could be a risk factor for thrombosis [81, 84]. They further provide evidence that HCMV infection might be involved in vascular disease.

enhance adverse clinical outcomes. Because some pathogens have been identified in atherosclerotic plaques, it has been hypothesized that they may indeed precipitate vascular inflammation, either by persistent infection [70, 87– 89] or by immune-mediated injury [24, 90–92]. An infectious theory of atherosclerosis was proposed by Benditt and Benditt [87] and further supported by Fabricant et al. [70]. Both, antigens and nucleic acid sequences of HCMV have been detected in SMCs from carotid artery plaques [93]. HCMV DNA was more often detected in arterial samples from patients with atherosclerosis than in control subjects [94]. Similarly, a higher prevalence of HCMV antibodies was observed in patients undergoing vascular surgery for atherosclerosis and significantly greater HCMV antibody titers were detected suggesting that the virus may have a role in the pathogenesis of atherosclerosis [95]. Indeed, one third of the atherosclerotic lesions obtained by coronary atherectomy harbor HCMV DNA sequences [94]. SMCs grown from such lesions express immediate early proteins IE84, one of the immediate early proteins of the virus that binds and inhibits p53 [96]. Inhibition of p53 by the virus is held responsible for the enhanced proliferation of SMCs and impaired apoptosis, either of which may contribute to restenosis [96]. It has also been reported that infection with HCMV is a strong independent risk factor for restenosis after coronary arterectomy [97]. In addition, a graded relation exists between the odds of intimal-medial thickening

HCMV and atherosclerosis A considerable number of epidemiologic studies suggest that infectious pathogens could predispose patients with cardiovascular diseases to atherosclerosis and that they

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and the levels of HCMV antibodies [98]. Importantly, the observed relation still remains significant after adjustment for the main cardiovascular risk factors. Moreover, there is also a significant correlation between high anti-HCMV IgG antibody titer and coronary artery disease [99]. Similarly, high anti-HCMV IgG antibody titers seem to predict postcoronary balloon angioplasty restenosis [99]. Also seropositivity for both Chlamydia pneumonea and HCMV infections was found to be associated with premature myocardial infarction even after adjustment for coronary risk factors and socioeconomic status [88]. Additional evidence for the possible link between HCMV and atherosclerosis comes from studies of arterial SMCs derived from patients with severe atherosclerosis [93]. Tissue samples from patients infected with HCMV revealed the presence of infected cells in virtually all organs [100] showing that HCMV can infect a wide variety of cells, among them monocytes/macrophages, fibroblasts, SMCs, and ECs [100]. However, the mechanisms of HCMV dissemination remain unclear. The cells of the myeloid lineage, specifically monocytes, are thought to have a central role in this process [30, 101–106]. First, monocytes containing viral DNA have been found in patients with cell-associated viremia [105, 106]. Second, HCMV-infected macrophages, as differentiated counterparts of circulating monocytes, have been detected in various organs of symptomatic and asymptomatic hosts [106–109]. Third, animal studies with the related murine and rat CMV b-herpes viruses showed that during primary infection infected monocytes are associated with the systemic spread of the virus through the blood [101, 110, 111]. On the other hand, a caveat to monocyte participation in HCMV dissemination are results suggesting that monocytes are only abortively infected [103, 105, 106, 112, 113]. However, HCMV infection of monocytes results in cellular activation, induction of cellular motility, and enhanced monocyte migration through endothelium [114, 115]. Interestingly, HCMV-infected peripheral blood monocytes, which at the time of infection were nonpermissive for viral replication, drove their differentiation into macrophages that then became permissive for replication of the originally present virus [114]. These studies suggest that the primary infection activates monocytes, promotes their migration into surrounding tissue and their differentiation into permissive macrophages, which in turn enables systemic viral spreading (Fig. 1).

HCMV infection and antibody titers Effective viral vaccines could induce antibodies that block viral entry into host cells. Entry of laboratory strains of CMV into fibroblasts is commonly used as an in vitro model

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for studying viral entry and antibodies that block it [116, 117]. Such studies revealed that critical epitopes for neutralizing antibodies reside in glycoproteins gB and gH. In addition, up to a half of the neutralizing activity of human immune sera seems to target epitopes within gB [118]. Two vaccines, the live attenuated Towne vaccine and the gBbased gB/MF59 protein subunit vaccine, induce titers of neutralizing HCMV antibodies in human subjects although to a lower extent than natural HCMV infections. Therefore, these vaccines might provide protection similar to that associated with natural seropositivity [119]. The Towne vaccine and the gB/MF59 subunit vaccine induce epithelial entry-specific neutralizing activities, which are on average 28-fold (Towne) or 15-fold (gB/MF59) lower than those observed following a natural infection [120]. Further, human immune sera and HCMV-hyperimmune globulins have on average 48-fold higher neutralizing activities in epithelial cells versus fibroblasts [120]. On the basis of these findings, it was suggested that a natural HCMV infection elicits neutralizing antibodies that are specific for the epithelial cell entry. As this activity could not be abolished with recombinant gB, it was proposed that the efficacy of a HCMV vaccine may be enhanced by induction of epithelial entry-specific neutralizing antibodies [120]. Beside antibody treatment against viral gB and gH, recently the UL131A–128 locus of the HCMV genome has been shown to be indispensable for the infection of ECs [121], as well as epithelial cells [38]. Particularly, the virion glycoprotein complex gH–gL–pUL131A–pUL130– pUL128 has been shown to be required for infection of both endothelial and epithelial cells, whilst the glycoprotein complex gH–gL–gO is required for infection of human fibroblasts [38]. Evidence was provided showing that gH/ gL/UL128–131 is necessary for efficient viral entry into epithelial cells of oral and genital mucosa [122]. The short peptides from UL130 and UL131 elicited high neutralizing antibody titers in rabbits, that can neutralize viral entry into epithelial cells derived from relevant tissues [122]. Therefore, it was suggested that single subunits or peptides may be sufficient to elicit potent epithelial entry neutralizing responses and that such antibodies may have the potential to provide protection by blocking initial mucosal infection [122]. On this background, a striking difference was observed when the neutralizing activity of human sera was analyzed from subjects with primary and reactivated HCMV infections on endothelial and fibroblast cells [123]; the neutralizing antibodies were markedly more efficacious when measured in endothelial and epithelial versus fibroblast cells. In addition, these antibodies were shown to inhibit HCMV plaque formation and virus transfer from HCMV-infected cells to leukocytes, thus displaying a similar range of protective activity than the neutralizing antibody response during a natural infection [123].

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Thus, antibodies to pUL131A, pUL130 and pUL128 are prime candidates for a differential neutralizing activity. Indeed, the anti-viral activity of anti-pUL131A/-pUL130/pUL128 monoclonal antibodies has been recently demonstrated [124].

Current therapies for treatment of HCMV-related diseases The current therapeutic regimens for the treatment of HCMV-related diseases include drugs such are ganciclovir, foscarnet, and cidofovir. Ganciclovir, together with its oral analog valganciclovir, is a guanosine analogue that acts as a DNA terminator during virus replication after phosphorylation by the CMV UL97 kinase. Ganciclovir or valganciclovir allow treatment of congenital HCMV disease [125]. In addition, ganciclovir has been tested in randomized controlled trials in transplant and HIV-infected individuals [126, 127]. It should be noted, that ganciclovir can be applied locally, for example, to the eye of patients with retinitis [126]; and neutropenia is the predominant systemic side effect of ganciclovir [128, 129]. Foscarnet is as effective as ganciclovir, but its main side effects are renal toxicity and electrolyte imbalance [130]. Cidofovir proved to be effective in the treatment of HCMV-related retinitis [126]. However, no randomized trial has been conducted in transplant recipients [131]. High dose acyclovir or valacyclovir have been demonstrated to reduce posttransplant cytomegalovirus disease in renal transplant patients [132, 133]. However, a recent study reported acute renal failure as a consequence of acyclovir therapy [134]. Although, antiviral drugs have been proven beneficial, drug resistance emerges as inconvenient setback [135]. Mutations affecting the virus UL97 kinase or, less often, the virus DNA polymerase can cause ganciclovir resistance [135]. Since foscarnet and cidofovir do not require phosphorylation by UL97, their resistance arises by mutations of the DNA polymerase gene [135]. In the past years, experimental therapies aimed at the treatment of HCMV infections emerged. Several new antiviral compounds are currently in phase II clinical trials. These include CMX001, a lipid derivate of cidofovir [136], and AIC246 aimed to block a late step in HCMV replication [137]. Maribavir, the UL97 kinase inhibitor, exhibited low toxicity and showed efficacy at least in one controlled trial [138]. In phase III clinical trials, maribavir prevented HCMV disease when started after engraftment [139]. In addition, there are approved drugs that possess anti-HCMV activity in vitro. These include leflunomide, which inhibits a late step in virion assembly [140]. Leflunomide has been used in salvage situations for treatment of HCMV disease

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[140]. However, we are not aware of any trials that would assess its efficacy and toxicity either as monotherapy or in combination. With regard to HCMV-specific and pooled immunoglobulins, prophylaxis had little success in transplant recipients [141, 142]. However, a recent uncontrolled trial suggested that this approach might be useful as a prenatal therapy aimed to prevent infection and disease in infants whose mothers acquired HCMV during pregnancy [143].

HCMV infection in transplant recipients HMCV seroprevalence is increased with age indicating that the HCMV infection could be acquired at any time during life [144]. In early childhood, acquisition of infection can happen via saliva in the family or day care surrounding. During the later stages of life, HCMV is frequently transmitted sexually, but also via saliva (e.g., from infected children), and blood transfusions [145]. Primary infection is commonly asymptomatic, but may also be clinically manifested as a mononucleosis-like illness. Occasionally, infection could cause pneumonia or gastrointestinal disease [146]. However, in immunocompromised individuals such as patients receiving immunosupressive therapy due to organ transplantation, HCMV infection accounts for a high morbidity and mortality rate. In addition, transplant organ rejection is highly increased in infected patients [18, 21–23]. The most important pretransplant risk factor for HCMV disease is the serological status of the donor and recipient. For example, during hematopoetic stem cell transplant (HSCT) procedure, seropositive recipients are at the highest risk, followed by HCMV-seronegative recipients receiving stem cells from HCMV-seropositive donors (D?/ R- patients). Seronegative recipients of stem cells from seronegative donors have the lowest risk to acquire primary infection [145]. Otherwise, the stem cell source and standardized conditions only minimally contribute to the risk of acquiring infection and HCMV-related disease. An exception is umbilical cord blood transplantation associated with reactivation and high disease rate in the absence of antiviral prophylaxis [147]. Among HSCT recipients, HCMV-related pneumonia is the most severe and feared health complication. Even despite the treatment, the mortality of such patients remains relatively high [148]. HCMV gastrointestinal disease is the second most frequent complication affecting upper and lower tracts. Other complications such as retinitis, hepatitis, and encephalitis occur infrequently. Nowadays, the use of preventive antiviral therapy or prophylaxis is standard care in HSCT recipients [149]. These strategies have reduced the incidence of HCMV disease from 25 to 30% in seropositive recipients to 5% during the first 3 months. However, late

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virus-related diseases may occur requiring continuous monitoring of high-risk patients [149]. In solid organ transplant (SOT) recipients HCMV can cause a febrile syndrome with leukopenia and/or transaminitis, as well as other end organ diseases. The highest risk of HCMV disease occurs in D?/R- patients. In contrast to HSCT recipients, reactivation disease is less common in seropositive SOT recipients. There seem to be proneness of clinical disease manifestations for the transplanted organ, most likely caused by minor human leukocyte antigen (HLA) mismatches. In this clinical setting, a HLA mismatch is a plausible trigger promoting local HCMV reactivation and replication [150]. Therefore, the end-organ disease manifestations differ according to the type of organ transplantation [151]. For example, after renal transplantation, new-onset diabetes mellitus after transplantation (NODAT) is an important complication. NODAT is responsible for increased mortality mainly related to cardiovascular events and infections. It has also been identified as an independent risk factor associated with graft loss [152]. It was concluded cyclosporine A is preferred over tacrolimus as the treatment of choice. In addition, a steroid-free regimen or early steroid withdrawal is encouraged to reduce the risk of NODAT [152]. In pancreas transplant patients, a CMV mismatch (D?/ R-) was identified as an additional risk factor for delayed graft function (DGF) occurrence. In this particular cohort, DGF does not seem to affect graft survival [153]. In lung transplantation, HCMV has been associated with producing direct, i.e., organ and systemic infection/disease, as well as indirect effects including acute rejection and chronic allograft dysfunction that limited the success. In this clinical setting, the specific cellular immune response plays a crucial role in supporting viral replication. A delayed or reduced response can represent the pathogenic basis for recurrent infections that may become symptomatic [154, 155]. Sester et al. reported a correlation between the low levels of specific T-cell reactivity and the frequency of infectious episodes [156]. The differences in T-cell reactivity within the groups of heart, kidney and lung transplant recipients were associated with varying doses of immunosuppressive drugs, as exemplified for calcineurin inhibitors that dose-dependently reduce specific T-cell reactivity [156]. The same authors concluded that monitoring the CMV-specific effects on CD4? T cells may allow prediction of long-term disease susceptibility and may contribute to an improved management of HCMV complications after lung transplantation [156]. Furthermore, the lack of a specific cellular immune response in lung transplantation may lead to the onset of potentially deadly lung disease as evidenced in a patient who developed HCMV pneumonia and eventually died, whereas infectious episodes were favorably resolved in responder

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patients [157]. Viral replication at non-pathologic levels may not affect the response of T cells as evidenced by the significantly different viral loads in the bronchoalveolar lavage fluid from responders and nonresponders [157]. Furthermore, mannose-binding lectin deficiency is associated with HCMV reactivations and a longer overall survival, but not with the development of bronchiolitis obliterans syndrome [158]. Factors associated with the development of CMV infection were validated in patients following 12 months after SOT [159]. A positive donor serostatus was associated with an increased risk of HCMV infection in recipients with both negative and positive serostatus. Antiviral drugs, such as cidofovir, foscarnet, ganciclovir and valganciclovir, were found to reduce the risk of HCMV infection, but only during the period of their active use [159]. In a retrospective study, valganciclovir was effective and safe for long term HCMV prophylaxis when administered before and 4 months after heart and lung transplantation [160]. These data demonstrate that transplant patients are at higher risk to be affected by HCMV infection. Hence, more attention should be paid to the analysis of the role of viral infection in acute or chronic rejection. Particularly, studies monitoring viral infection a year or later after transplantation, when antiviral prophylaxis is discontinued, are necessary because of the possible development of lateonset HCMV disease [154, 155]. In addition, evaluation of HCMV-specific cellular immune responses may complement virologic monitoring, helping to identify recipients at risk of developing organ infection or disease.

Conclusion In this review we provide an overview of the contribution of HCMV to vascular pathology with particular focus on atherothrombosis. Whether or not infectious agents are involved in the pathogenesis of atherosclerosis has been a matter of intensive discussion for the past two decades. Although there is no definite proof of a causal role of HCMV in atherogenesis, there is an ever growing body of evidence implying an important role of this virus in vascular pathology. All evidence so far supports the notion that in already atherosclerotic vasculature HCMV triggers an inflammatory response, which could in turn potentiate the severity of the disease, particularly in immunocompromised hosts. On the other hand, in immunocompetent hosts, HCMV infection may be successfully controlled by joint actions of the innate and adaptive immune system. However, the ability of HCMV to establish a life-long persistent infection poses an ubiquitous threat for the host. Weakening the immune system by any means could initiate

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reactivation of HCMV and onset of inflammatory responses. In addition, transplant patients are at greater risk of acquiring HCMV-related disease due to susceptibility to primary infection or virus reactivation. It is clear that future studies should focus on investigating the potential implications of these findings in terms of indirect effects of viral infection/disease, such as onset of acute or chronic rejection. In particular, studies monitoring viral infection beyond 1 year after transplantation, when antiviral prophylaxis is discontinued, are necessary because the development of late-onset HCMV disease is possible. Evaluation of HCMV-specific cellular immune responses by viroimmunologic monitoring may complement virologic monitoring, thus helping to identify transplant recipients that are at increased risk of developing organ infection or disease. Since it has been acknowledged as an important mediator of vascular pathology, HCMV has been identified as one of the key targets for novel therapeutic interventions to minimize irreversible tissue damage associated with thrombosis and atherosclerosis. Future studies should focus on the mechanisms by which HCMV exerts its inflammatory effects in atherothrombosis, as well as on novel strategies aimed to prevent clinical complications of atherosclerosis and thrombosis. Acknowledgments This work was supported by grants: Deutsche Forschungsgemeinschaft, Si 285/7-1 (to Tatiana Syrovets and Thomas Simmet), and Serbian Government Research Grants, no. 173033 (to Esma R. Isenovic´) and no. III41028 (to Milan Popovic´).

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