Pathobiology

The prevailing concept is the one which dysfunctional endothelial cells plays the key pathobiological role in PH [20]. However, to the present day, the pathogenesis of intimal vascular lesions remains mostly undetermined. It is conceivable that these lesions result from injuries to the endothelium, followed by activation of smooth muscle cell migration, extracellular matrix deposition, and endothelial cell proliferation. As mentioned, it is also possible that these lesions occur due to organization of microthrombi. In animal models, only the association of the alkaloid monocrotaline (MCT) and high shear stress due to shunting between the systemic and pulmonary circulations leads to marked intima thickening by smooth muscle cells and severe PH in rats [21]. The intimal cells express smooth muscle cell markers, and fibronectin [19].

We have proposed that plexiform lesions represent a process of misguided angiogenesis based on the findings of expression of vascular endothelial growth factor (VEGF), its receptors 1 (flt) and 2(kdr), and hypoxia inducible factor (HIF)-1α and β [22]. The finding of monoclonality of endothelial cells in plexiform lesions in IPAH, but not in similar lesions in PH associated with congenital heart malformation, suggests that these lesions might arise from mutations in tumor suppressor genes [23]. Somatic loss of expression of transforming growth factor β receptor 2 ((TGFβ-R2) and the propapoptotic Bax, potentially due to microsatellite instability, is also documented in IPAH plexiform lesions [24]. Heterozygous germline mutations of bone morphogenetic protein receptor 2 (BMPR2) (a component of the TGF-β-related family) is found to underlie up to 60% of cases of familial IPAH [25;26]. BMPR2 expression is documented in plexiform lesions [12] and in remodeled pulmonary arteries in IPAH lungs, predominantly in endothelial cells. However, our studies reveal that the cells in the central core of plexiform lesions lack the expression of TGF-β receptor 2, TGF-β receptor 1 and their signaling smad(s) 2,1 (which shares common epitopes with smads 5 and 8), 3 and 4, including the phosphorylated smad 1/5/8 and 2. The absence of expression of these phosphorylated smads is the best indication that there is no TGF-β (via smad 2 or smads 1/5/8) or BMP (via smads 1/5/8) signaling in these cells [27]. Loss of cytostatic signaling from TGF-β would allow the plexiform cells to abnormally proliferate. Notwithstanding the evidence of preserved BMPR expression and signaling in IPAH endothelial cells, recent studies indicate that BMP signaling protects against endothelial cell apoptosis in vitro [28]; the loss of this protection by germline mutations in BMPR2 would thus favor enhanced susceptibility to apoptosis of lung endothelial cells.

The potential role for early endothelial cell apoptosis in the pathogenesis of uncontrolled proliferation of pulmonary endothelial cells was first documented in the rat model of severe pulmonary hypertension caused by the combination of VEGF receptor blockade with SU5416 and chronic hypoxia [29]. Moreover, the role of endothelial cell apoptosis in the pathogenesis of PH was also extended to the monocrotaline model [30]. Initial endothelial cell apoptosis might favor the emergence of apoptosis-resistant endothelial cells, with potential for uncontrolled proliferation [29;31].

VEGF [22], endothelin-1 [32], and survivin [33;34] are among the factors present in plexiform lesions that may enhance endothelial cell and smooth muscle cell proliferation or decrease vascular cell apoptosis. These lesions have decreased expression of anti-remodeling mediators such as nitric oxide synthase [35] and prostacyclin synthase [36], and tumor suppressors, such as caveolin-1[37]. These phenotypic characteristics lead us to propose that the plexiform lesions have characteristics in common with neoplasms [38]. As discussed below, there is emerging evidence that viral factors may play a role in endothelial cell proliferative lesions in severe PH.

A summary of the molecules that may contribute to lesion formation, their expression, and the signaling or metabolic pathways involved, as well as their potential target in the vasculature are shown in Table 3.

Pathobiology

The expansion of the tunica media in pulmonary arteries in PH occurs by a combination of hypertrophy and hyperplasia. Smooth muscle cell hypertrophy together with accumulation of extracellular matrix, including thicker elastic laminae, accounts for most of medial thickening in PH [43]. Smooth muscle cell hyperplasia probably plays a minor role in medial thickening than cell hypertrophy or extracellular matrix deposition, since it is experimentally shown that the proliferation index of smooth muscle cells doubles in large pulmonary arteries in hypoxic rats, while those cells lining intraacinar arteries do not show evidence of [³H]-thymidine uptake [44]. A similar finding is observed in PH caused by monocrotaline in rats [45]. The apparent predominant role of smooth muscle cell hypertrophy over hyperplasia in medial thickening is also supported by human PH studies [19;46;47]. Based on the hypoxia model of PH, the appearance of smooth muscle cells in nonmuscularized precapillary arteries occurs by hypertrophy and metaplasia of potential precursor cells, i.e., pericytes or intermediate cells (pericytes-smooth muscle cells) [44]. A careful examination of the smooth muscle cell phenotypes in the tunica media of neonatal bovine pulmonary arteries reveals the existence of substantial cellular heterogeneity, with clusters of fully differentiated smooth muscle cells interspersed with cells negative for most smooth muscle cell markers [48]; such a cellular heterogeneity probably exists in the different forms of human PH as well, though a systematic dissection of these phenotypes has not been performed thus far. In spite of these data, most of the present day studies addressing the potential role of genetic mutations or alterations in ion channels have focused on smooth muscle cell proliferation, as noted below.

The mechanisms underlying the thickening of the pulmonary vascular medial layer have been linked mostly to cell proliferation and, more recently, to inhibition of cell apoptosis. The identification of mutations in BMPR2 led to several in vitro studies aimed at relating smooth muscle cell proliferation to abnormal BMPR2 signaling. IPAH smooth muscle cells isolated from proximal arteries (i.e., elastic vessels, >500 μm in diameter) exhibit decreased inhibitory effect on cell proliferation mediated by TGF-β1 or BMP-4 when compared with smooth muscle cells isolated from normal human pulmonary arteries [49]. Of note, these altered responses are not due to abnormal expression of smad signaling or ligand binding to its receptors in IPAH smooth muscle cells. Similar studies were extended to more peripheral smooth muscle cells (i.e., from arteries less than 2 mm in diameter-(that are still elastic arteries) obtained from normal pulmonary arteries, which undergo cell proliferation and protection against apoptosis when exposed to BMP-4, a ligand for BMPR2. Therefore, loss of function mutations in MPR2 would be predicted to cause smooth muscle cell arrest and increased cell death [50], i.e., paradoxically opposite to that predicted to occur in familial IPAH in which mutations would facilitate smooth muscle cell growth and remodeling. Given these somewhat discrepant results [50], the evidence of reduced expression of BMPR2 found in IPAH alveolar septa [12], and the finding of decreased levels of the activated form of smad 1 (the signaling smad for BMPR2) in smooth muscle cells of muscular pulmonary arteries in IPAH, it remains unclear how BMPR2 mutations either can cause or trigger the disease. Indeed, when compared with wild type mice, heterozygous mice lacking a single copy of BMPR2 have no pulmonary vascular phenotype at baseline or under chronic hypoxia [51], but show moderately increased pulmonary artery pressures and minimal remodeling when stressed with intratracheal delivery of a 5-lipoxygenase expressing vector [51].

The role of serotonin (5-hydroxytryptamine) in the growth stimulation of pulmonary artery smooth muscle cells has been intensively studied as a mechanism of medial remodeling in PH and a potential modifier gene to the familial IPAH. Serotonin is internalized in smooth muscle cells after binding to serotonin transporter (5-HTT) or to its receptors 5-HT_1B-R, 5-HT-_2A R, 5-HT₇ -R, and 5-HT_2B -R, when it promotes vasoconstriction, cell growth, and enhancement of hypoxia-induced remodeling and PH [52]. Vascular smooth muscle cells in IPAH lungs express higher levels of 5-HTT [53] or 5-HT_2B R [52] by immunohistochemistry, and undergo enhanced cell proliferation when treated in vitro with serotonin compared with normal smooth muscle cells [53]. These findings are supported by the report that mice deficient in 5HTT or 5-HT-_2B are protected against PH caused by hypoxia alone [52] or hypoxia combined with the anorexigen dexfenfluramine [54], respectively. Of interest is the observation that increased serotonin levels affect signaling pathways downstream of mutated BMPR2, as serotonin infusion enhances normoxic and hypoxic pulmonary pressures and vascular remodeling in BMPR2 heterozygous mice as compared with wild type mice [50].

Despite evidence supporting a loss of function for the TGF-β family signaling, particularly in endothelial cells of IPAH, TGF-β may produce gain-of-function alterations underlying medial smooth muscle cell growth and adventitial fibroblast activation. TGF-β isoforms 1, -2, and -3 are expressed in hypertensive pulmonary arteries [55], and could signal via activation of smad 2 or 3 by serine phosphorylation, whose expression was documented in pulmonary vascular smooth muscle cells [27]. Recent evidence implicates PDGF in the pathogenesis of both monocrotaline- and chronic hypoxia-induced PH [56]. Monocrotaline treatment or chronic hypoxia exposure leads to increased PDGF receptor (PDGFR)-β expression and phosphorylation and activation of ERK Map kinase in rats. The findings of positive response of hypertensive animals to Gleevac™, also an inhibitor of PDGFR signaling, and the evidence of increased expression of PDGFR-β and phosphorylated PDGFR-β in IPAH lungs, led the authors to translate their finding by treating an IPAH patient with Gleevac™, with promising early results [57].

The extracellular matrix is both a component of the thickened pulmonary vascular media and a regulator of smooth muscle cell growth. Tenascin, a glycoprotein involved in lung and vascular morphogenesis, is strongly expressed in remodeled intima and medial layers of human hypertensive pulmonary arteries [58]. Experimentally, products of enhanced elastolytic or proteolytic activity in pulmonary arteries can promote clustering of integrin receptors, which activate tenascin and epidermal growth factor (EGF) receptors, thus leading to smooth muscle cell proliferation [59]. These findings are pertinent to the monocrotaline model of PH [60;61], while pulmonary vascular remodeling in hypoxic pulmonary hypertension is improved by the action of metalloproteases [62]. Based on studies using cultured human smooth muscle cells isolated from elastic arteries from IPAH patients, it appears that Tissue Inhibitor of Metalloprotease-1 (TIMP-1) and metalloprotease (MMP)-2 are increased in expression; only the finding of MMP-2 enhanced expression is confirmed in situ in elastic pulmonary arteries in IPAH lungs [63]. It is unclear whether a similar pattern is present in distally remodeled pulmonary arteries; however, endothelial cells express moderate/intense immunohistochemical expression of MMP-2, while myofibroblasts display low levels of this extracellular protease [64]. Membrane type-1-MMP was also expressed in endothelial and myofibroblastic cells of concentric and plexiform lesions. These results and those in the monocrotaline model suggest that the inhibition of elastases or MMPs might be beneficial in IPAH as demonstrated in the monocrotaline model of PH [65], potentially leading to the apoptosis of smooth muscle cells.

It is becoming clear that the ultimate fate of vascular smooth muscle cells in PH is determined by their resistance to apoptosis. In fact, ‘apoptosis-resistance’ might play a central role in both the endothelial- and smooth muscle cell-based pulmonary vascular lesions [66], since IPAH lungs have lower number of apoptotic cells than normal or emphysematous lungs [67]. As growth signals originated by PDGF, TGF-β, EGF, serotonin, and extracellular matrix proteins are interrupted in animal models of PH, pulmonary arteries undergo de-remodeling associated with apoptosis of pulmonary artery smooth muscle cells [65]. Targeting apoptosis of the hypertrophic smooth muscle cells might represent a more viable approach towards treatment.

Recent studies of K⁺ channel activity have provided a novel insight of the lack of proapoptotic signals in IPAH smooth muscle cells. An exciting and complementary paradigm based on the interplay between K⁺ channels and apoptosis in pulmonary artery smooth muscle cells has emerged based on the demonstration of activation of K⁺ channels causes cytochrome C release from the mitochondria and water efflux from the dying cells [68]. Conversely, inhibition of K⁺ channels causes cell depolarization, enhances contractility, and decreases apoptosis of pulmonary artery smooth muscle cells. One potential mechanism linking this paradigm to BMPR2 mutations is the finding that BMPR2 activation upregulates K⁺ channels [69] and causes apoptosis of normal pulmonary artery smooth muscle cells, but not of cells from patients with IPAH [70]. McMurtry et al provide recent additional evidence that mechanisms akin to cancer operate in pulmonary vascular remodeling [34]. Survivin protects cancer cell against apoptosis by inhibiting caspase activation and apoptosis inducing factor [71]. Not only do pulmonary artery smooth muscle cells in IPAH lungs express higher levels of the antiapoptotic survivin, but monocrotaline-induced pulmonary vascular remodeling requires survivin expression. Indeed, transduction of a functionally deficient survivin in monocrotaline-treated lungs prevented pulmonary vascular remodeling and, when administered after monocrotaline treatment, it enhances apoptosis of pulmonary artery smooth muscle cells and reduces pulmonary artery pressures. This mutant survivin increases levels of K⁺ channel activity and leads to depolarization of mitochondria with enhanced cytochrome C release.

Pathobiology

It is apparent that the adventitial fibroblasts react promptly to vascular stresses, notably those caused by hypoxia, high-perfusion flow models, and, to a lesser extent, the alkaloid monocrotaline treatment [72]. Fibroblasts undergo a marked increase in cell proliferation reaching 11-fold increase in labelling index in hilar vessels, at day 3 of chronic hypoxia exposure [44]. In the monocrotaline model, proliferation rate of adventitial fibroblasts reaches a 5-fold increase at day 21 [45]. These findings underscore the powerful abilities of adventitial fibroblasts to undergo proliferation, one of the most characteristic properties of these cells. Moreover, adventitial fibroblasts can: 1. either differentiate into smooth muscle cells and migrate into the remodeled media or; 2. trigger smooth muscle cell proliferation by secreting growth factors; 3. allow for the recruitment of inflammatory and bone marrow progenitor cells; 4. create a ‘vasculogenic and angiogenic’ niche for expansion of newly-formed vessels (vasa vasorum). It is apparent that adventitial fibroblasts might represent heterogeneous clusters of cells, like those found in the medial smooth muscle cells [72]; this biological heterogeneity is readily apparent in the neonatal bovine model of hypoxic PH. A potentially important finding is the contribution of circulating fibrocytes (precursor cells sharing the expression of the fibroblast marker α1-collagen and peripheral leukocyte markers CD45, CD34, CD11b, and CD14) to experimental hypoxic PH [72]. Adventitial fibroblasts sense alterations in their redox status, with the ensuing activation of cell growth, cytokine release, and generation of oxidants via activation of NADPH oxidase [72]. The redox regulation of adventitial cell function is supported by the identification of markers of oxidative stress in remodeled pulmonary arteries of IPAH lungs [73].

Pathobiology

Little is known about the functional alterations of endothelial, smooth muscle, and adventitial fibroblasts in venous PH. Pulmonary veins are difficult to identify in lung sections, given the fact that they are located in the midst of the alveolated tissue. Only vein segments present in interlobular septa (i.e., invaginations of pleural connective tissue containing veins and lymphatics) can be more easily recognized. It is apparent that the development of endothelial cell markers unique to pulmonary veins would aid in the proper assessment of the contribution of endothelial cell dysfunction in venous pulmonary hypertension. The same limitations apply to venous smooth muscle cells and fibroblasts.

Pathobiology

While perivascular inflammation has limited value in the pathological work up of cases of PH, it is presently one of the most promising areas of investigation in the disease. Inflammatory cells may interact with viral factors, which have emerged as potential etiological/pathogenetic agents in PH.

There is compelling evidence of global immunological alterations in IPAH patients [76;77] and PH occurs in the setting of profound immune deregulation underlying HIV infection and collagen vascular diseases. The recognition of an inflammatory component in PH [16;74] supports the investigation of expression of cytokines that might potentially drive perivascular inflammation and thus contribute to the disease. Remodeled pulmonary arteries express IL-1, IL-6, and PDGF in infiltrating inflammatory cells [78;79], the chemokine RANTES (acronym for regulated upon activation, normal T-cell expressed and secreted), an important chemoattractant for monocytes and T cells [80], and the macrophage inflammatory protein-1α (MIP-1 α) [76]. Lungs of IPAH patients have increased expression of fractalkine, a chemokine involved in T cell trafficking and monocyte recruitment, and their circulating CD4 and CD8 T-cells have higher levels of the fractalkine receptor CX3CR1 when compared with controls or samples of patients with thromboembolic PH [81].

Inflammatory cells infiltrating remodeled pulmonary arteries may include subpopulations of vascular precursor or early-progenitor cells, also potential contributors to pulmonary vascular remodeling in PH. Pulmonary arteries in PH caused by chronic hypoxia contain an infiltrating subpopulation of fibrocytes, identified by the expression mononuclear cell markers CD45, CD11b, CD14, and the fibroblast marker α1-procollagen. About 15% and 20% of these cells also undergo proliferation and express smooth muscle α-actin, respectively [82]. These studies also document that depletion of circulating monocytic cells alleviates pulmonary vascular remodeling caused by chronic hypoxia. Endothelial cell precursors may play a beneficial role in PH since their administration to monocrotaline-treated rats has dramatic healing effects in remodeled pulmonary arteries, notably when transfected with the endothelial nitric oxide synthase gene [83]. The potential for either deleterious or beneficial effects from manipulations that increase the progenitor cell pool underscores the need for careful and detailed studies in the human disease, prior to embarking on clinical trials.

Viral infection may disrupt normal immunoregulatory and homeostatic cellular pathways, which result in endothelial or smooth muscle cell injury and activate inflammation. Most of the pathways involved in virus pathogenesis converge on either pro-survival or pro-angiogenic signals, the same signals associated with severe PH.

The important role of inflammation is further highlighted in cases of PH where a viral etiology can be identified. For HIV-related pulmonary hypertension, the first clinical report of an association between infection and the development of lesions appeared in 1987 [84], followed by other reports [85–87] but with no evidence for the presence of the virus in PH vascular lesions. As PH is frequently diagnosed when it is advanced, the incidence of PH in HIV-infected patients is likely underestimated, although a recent report demonstrates a high prevalence of PH in HIV-infected children [88]. BMPR2 mutations are not required for severe PH to occur in HIV-infected patients, yet the vascular lesions in the lungs from HIV-infected [86] patients are identical to those with familial PH and sporadic IPAH with BMPR2 mutations.

Some studies showed no correlation between viral load and right heart changes [89;90]. However, a case report [91] and a recent unpublished observation showed that viral load control with HARRT therapy can be associated with an improved clinical outcome (M. Humbert, personal communication). Furthermore, bosentan, an endothelin receptor antagonist has been successfully used in some HIV-PH patients [92], suggesting that shear stress contributes to the disease independently of the viral load.

In the lung, HIV-1 primarily infects macrophages providing a potential reservoir not only for the transmission of the virus to circulating T-cells, but also a source for localized viral proteins such as Nef, Tat,and gp120, all of which may have direct effects on innocent by-stander cells. The chronic exposure to viral products in the lung, a deficiency in regulatory T cells, and an altered production of chemokines/cytokines, may all contribute to pulmonary vascular dysfunction, with endothelial cells being particularly sensitive target.

The HIV Nef (for negative factor) protein is found in plexiform lesions of macaques infected with a chimeric virus containing the simian immunodeficiency virus (SIV) backbone with the human immunodeficiency virus Nef (in place of SIV nef) [93]. Nef is also present in endothelial cells of HIV infected patients with PH (Figure 7) [93].

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Figure 7

HIV-1 Nef localization in HIV-1 associated PH. A. Normal precapillary vessels (methyl green). B. Localization of Factor VIII related antigen stained with Alexa Fluor 488 (green) and Nef stained with Vector Red. Note lack of expression of Nef in normal pulmonary arteries, resulting in a green signal in the endothelial cells (arrow). C. Markedly remodeled pulmonary artery with concentric/plexiform lesion (arrow, methyl green). D. Co-localization of Factor VIII related antigen (green) with Nef (red), resulting in a yellow signal (arrow). E. Plexiform lesion in HIV-1 associated PH (arrow, methyl green). F. Co-localization of Factor VIII related antigen (green) with Nef (red), resulting in a yellow signal (arrow) in endothelial cells of pulmonary arteries with mild remodeling (arrow). G and H. Lymph node of an SIV infected monkey, showing extensive expression of Nef (red in G and brown in H, arrows) in a hilar lymph node. (Reproduced from Marecki, J. C., et al. “HIV-1 Nef is Associated with Complex Pulmonary Vascular Lesions in SHIV-nef-infected Macaques.” Am.J Respir Crit Care Med. 174.4 (2006): 437-45, with permission.)

Nef is also present in endothelial cells of HIV infected patients with PH. These recent studies suggest that the viral protein may exert direct effects on cells not necessarily permissive for viral replication. Foci of mononuclear cells and ectopic lymphoid tissues characteristically found in regions adjacent to the lesions may be sources of this viral protein (Figure 5).

The Nef protein appears to be dispensable for viral replication in vitro, but is a critical virulence factor for pathogenesis and maintenance of high viral loads in vivo [94;95]. Nef is an N-terminus-myristoylated protein with a relative molecular mass of 27 kDa is found associated with cellular membranes and the cytoskeleton [96]. Myristoylation is essential for almost all the functions ascribed to Nef, including membrane localization within lipid raft microdomains. The localization and adaptor functions recruit signaling proteins to discrete regions in the membrane and affect T cell signaling pathways [97]. Proteins associated with a survival and pro-angiogenic phenotype in severe PH such as PI-3 kinase, MAP kinases, and a p21 kinase-2 are all recruited to the rafts by Nef [98–101]. In human monocyte-derived macrophages (MDMs), Nef activates the STAT1 pathway and the secretion of MIP-1, IL-1-α, IL-6, and TNF-α [102]. Extracellular Nef found in HIV patients (approximately 10 ng/ml) enters the vascular endothelium in vivo via CXCR4 [103]. Finally, Nef can be proapoptotic or pro-survival, depending on the context of expression and the particular cell type [104]. Thus, localization of Nef to the lipid rafts may be sufficient to trigger the changes associated with the endothelial cell expansion characteristic of plexiform lesions. On the other hand, a second hit such as infection with other viruses (e.g. gammaherpesviruses such as HHV8) or a genetic susceptibility may be necessary as well.

Human herpesvirus 8 (HHV8) is a gammaherpesvirus, also known as Kaposi’s sarcoma-associated herpes virus (KSHV) [105;106]. Evidence of HHV8 is found in a large percentage of plexiform lesions of PH patients examined in Denver, USA. suggesting for the first time that this virus was a contributing factor [107]. There are several pro-angiogenic or oncogenic genes present in its genome, including a viral IL-8 and a viral IL-6 both shown to play a role in IPAH. In addition, the genome encodes a seven-transmembrane-spanning G protein-coupled receptor (GPCR) with extensive sequence similarity to cellular chemokine receptors [108]. When expressed in NIH 3T3 fibroblasts, this gene increases their ability to grow in soft agar and to induce tumor formation in nude mice [109]. GPCR increases secretion of vascular endothelial growth factor and activation of the ERK1/2 (p44/42) mitogen-activated protein kinase signaling pathway [110]. Endothelial cells that express this gene become immortalized with constitutive activation of the VEGF-receptor 2 (KDR) [111]. In addition, this gene can cause KS-like lesions in nude mice [109], and over-expression within hematopoietic cells results in angioproliferative lesions, resembling those found in KS [112]. These are yet more examples of viral factors with the potential of altering cellular phenotype in the absence of viral replication. Nevertheless, in spite of their recognized angioproliferative potential and the initial association with plexiform lesions, several groups do not reproduce these results with HHV8 [113]. Studies of patients from a San Francisco clinic along with Japanese and German cohorts find no evidence of latent virus in the lesions or serum antibodies against viral antigens [114–117]. The discrepancy between groups may be a reflection of the methodology used to detect the virus or of regional (genetic/environmental) differences in the study population. Of note, latency-associated transcripts may be undetectable if the virus is going through a lytic replication cycle. In addition, serological tests for viral antibodies are notoriously difficult and in many cases, hard to interpret. Thus, further studies are necessary to address these questions.

PH represents one of the extrahepatic complications of Hepatitis C virus infection, with a prevalence of 1–5% [118]. In the majority of patients, portal hypertension precedes pulmonary hypertension [118;119]. The pathogenesis is also poorly understood, but the histological hallmarks are similar to IPAH. Whether these lesions are secondary to increased inflammatory cytokine production or to direct viral replication or to presence of viral products in the lung remain to be determined. As in HIV-mediated pulmonary hypertension, an associated immune dysregulation may trigger uncontrolled intrapulmonary angiogenesis.

Some case report studies of pulmonary hypertension patients showed infection of lung endothelial cells with other viruses like herpes simplex type 2 or cytomegalovirus. The fact that both belong to the viral family Herpesviridae raises the intriguing possibility that they may share some of the angioproliferative potential of HHV8. Nevertheless, since both patients were HIV-positive, it is difficult to dissect the contribution of each virus to the angioproliferation.