Mechanisms of cytomegalovirus-accelerated vascular disease: induction of paracrine factors that promote angiogenesis and wound healing.

Human cytomegalovirus (HCMV) is associated with the acceleration of a number of vascular diseases such as atherosclerosis, restenosis, and transplant vascular sclerosis (TVS). All of these diseases are the result of either mechanical or immune-mediated injury followed by inflammation and subsequent smooth muscle cell (SMC) migration from the vessel media to the intima and proliferation that culminates in vessel narrowing. A number of epidemiological and animal studies have demonstrated that CMV significantly accelerates TVS and chronic rejection (CR) in solid organ allografts. In addition, treatment of human recipients and animals alike with the antiviral drug ganciclovir results in prolonged survival of the allograft, indicating that CMV replication is a requirement for acceleration of disease. However, although virus persists in the allograft throughout the course of disease, the number of directly infected cells does not account for the global effects that the virus has on the acceleration of TVS and CR. Recent investigations of up- and downregulated cellular genes in infected allografts in comparison to native heart has demonstrated that rat CMV (RCMV) upregulates genes involved in wound healing (WH) and angiogenesis (AG). Consistent with this result, we have found that supernatants from HCMV-infected cells (HCMV secretome) induce WH and AG using in vitro models. Taken together, these findings suggest that one mechanism for HCMV acceleration of TVS is mediated through induction of secreted cytokines and growth factors from virus-infected cells that promote WH and AG in the allograft, resulting in the acceleration of TVS. We review here the ability of CMV infection to alter the local environment by producing cellular factors that act in a paracrine fashion to enhance WH and AG processes associated with the development of vascular disease, which accelerates chronic allograft rejection.

The role of angiogenic and wound repair factors during CMV-accelerated transplant vascular sclerosis in rat cardiac transplants.

Human cytomegalovirus (HCMV) accelerates transplant vascular sclerosis (TVS), a consequence of angiogenesis (AG) and wound repair (WR). While HCMV can be localized to TVS lesions, the low number of infected cells suggests a global effect on target tissues. We used microarray analysis followed by real-time-polymerase chain reaction (RT-PCR) in an RCMV-accelerated TVS rat cardiac transplant model to determine whether CMV activates host WR and AG factors. Dysregulated cellular genes in allografts from RCMV-infected recipients were compared to those from uninfected recipients and native hearts. We demonstrated that RCMV upregulates the genes involved in WR and AG, which was highest during the critical time of TVS acceleration (21-28 days). Using a standard in vitro AG assay, virus and serum-free supernatants collected at 48 h postinfection significantly induced endothelial cell (EC) migration, branching and tubule formation compared to supernatants from mock-infected cells. Supernatants from ultraviolet (UV)-inactivated RCMV-infected cells failed to induce AG, indicating that virus replication is required. Upregulation of WR and AG genes occurs during the critical period of CMV-accelerated TVS. Targeting these genes may prevent this process and improve allograft survival.

Kaposi's sarcoma: a model of both malignancy and chronic inflammation.

Kaposi's sarcoma (KS) is a complex cancer characterized by angioproliferative multifocal tumors of the skin, mucosa and viscera. KS lesions are comprised of both distinctive spindle cells of endothelial origin and a variable inflammatory infiltrate. There are four different epidemiological forms of KS: classic (sporadic), African (endemic), AIDS-associated (epidemic), and immunosupression-associated (iatrogenic). Although these various forms of KS have different environmental and immunological components, the development of each depends upon infection with Kaposi's sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV8). KSHV encodes an arsenal of gene products that induce cellular proliferation, transformation, cell signaling, cytokine production, immune evasion, antiapoptosis and angiogenesis. Yet, KSHV alone is insufficient to give rise to KS. The exact origin of the tumor cell (spindle cell), which is generally agreed to be a type of endothelial cell, remains elusive. Current evidence supports their derivation from lymphatic endothelium. However, both lymphatic and vascular endothelial cell types can be infected by KSHV in vitro, and recent studies suggest that this virus may reprogram the target cell, thus masking the cell's true origin. It is also possible that the original target cell is an uncommitted progenitor. In addition to the potentially neoplastic spindle cells, the KS lesion also contains dendritic cells, macrophages, plasma cells and lymphocytes. The presence of this admixed immune infiltrate has led to the suggestion that KS may result from reactive hyperproliferation induced by chronic inflammation, and that it is therefore not a true neoplasm. This review details the data that support KS as a model of both oncogenesis and chronic inflammation.

The insulin receptor is essential for virus-induced tumorigenesis of Kaposi's sarcoma.

Kaposi sarcoma (KS), a multifocal neoplasm of the skin that can spread to visceral organs, is the most prevalent malignant tumor in acquired immuno deficiency syndrome (AIDS) patients. KS-associated herpesvirus (KSHV or HHV8) is considered the primary etiological factor of this malignancy, as well as of primary effusion lymphoma and multicentric Castleman's disease. KS lesions are characterized by proliferating spindle cells of endothelial cell (EC) origin. The action of the insulin-like growth factor (IGF) system has been implicated in many malignancies, and recent data have demonstrated that the IGF-I receptor (IGF-IR) is required for in vitro growth of the KS-derived KSIMM cell line. To examine whether the IGF pathway is also involved in KSHV-mediated transformation of ECs, we examined the expression and function of the IGF system in KSHV-infected, immortalized dermal microvascular EC (E-DMVEC). The expression of the insulin receptor (IR) was strongly induced in latently infected E-DMVEC, whereas the expression levels of the IGF-IR remained unchanged. Gene knockdown of IR, but not IGF-IR, prevented the characteristic focus formation seen in KSHV-infected E-DMVEC. Similarly, treatment with the IR-specific small-molecule inhibitor HNMPA-(AM(3)) inhibited postconfluent growth. These data suggest a role for the IR, but not the IGF-IR, in KSHV-induced transformation of vascular ECs.

Endothelial cell- and lymphocyte-based in vitro systems for understanding KSHV biology.

Kaposi sarcoma (KS), the most common AIDS-associated malignancy, is a multifocal tumor characterized by deregulated angiogenesis, proliferation of spindle cells, and extravasation of inflammatory cells and erythrocytes. Kaposi sarcoma-associated herpesvirus (KSHV; also human herpesvirus-8) is implicated in all clinical forms of KS. Endothelial cells (EC) harbor the KSHV genome in vivo, are permissive for virus infection in vitro, and are thought to be the precursors of KS spindle cells. Spindle cells are rare in early patch-stage KS lesions but become the predominant cell type in later plaque- and nodular-stage lesions. Alterations in endothelial/spindle cell physiology that promote proliferation and survival are thus thought to be important in disease progression and may represent potential therapeutic targets. KSHV encodes genes that stimulate cellular proliferation and migration, prevent apoptosis, and counter the host immune response. The combined effect of these genes is thought to drive the proliferation and survival of infected spindle cells and influence the lesional microenvironment. Large-scale gene expression analyses have revealed that KSHV infection also induces dramatic reprogramming of the EC transcriptome. These changes in cellular gene expression likely contribute to the development of the KS lesion. In addition to KS, KSHV is also present in B cell neoplasias including primary effusion lymphoma and multicentric Castleman disease. A combination of virus and virus-induced host factors are similarly thought to contribute to establishment and progression of these malignancies. A number of lymphocyte- and EC-based systems have been developed that afford some insight into the means by which KSHV contributes to malignant transformation of host cells. Whereas KSHV is well maintained in PEL cells cultured in vitro, explanted spindle cells rapidly lose the viral episome. Thus, endothelial cell-based systems for studying KSHV gene expression and function, as well as the effect of infection on host cell physiology, have required in vitro infection of primary or life-extended EC. This chapter includes a review of these in vitro cell culture systems, acknowledging their strengths and weaknesses and putting into perspective how each has contributed to our understanding of the complex KS lesional environment. In addition, we present a model of KS lesion progression based on findings culled from these models as well as recent clinical advances in KS chemotherapy. Thus this unifying model describes our current understanding of KS pathogenesis by drawing together multiple theories of KS progression that by themselves cannot account for the complexities of tumor development.

Targeted inhibition of calcineurin signaling blocks calcium-dependent reactivation of Kaposi sarcoma-associated herpesvirus.

Kaposi sarcoma-associated herpesvirus (KSHV) is associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman disease. Reactivation of KSHV in latently infected cells and subsequent plasma viremia occur before the development of KS. Intracellular signaling pathways involved in KSHV reactivation were studied. In latently infected PEL cells (BCBL-1), KSHV reactivation in single cells was determined by quantitative flow cytometry. Viral particle production was determined by electron microscope analyses and detection of minor capsid protein in culture supernatants. Agents that mobilized intracellular calcium (ionomycin, thapsigargin) induced expression of KSHV lytic cycle-associated proteins and led to increased virus production. Calcium-mediated virus reactivation was blocked by specific inhibitors of calcineurin-dependent signal transduction (cyclosporine, FK506). Similarly, calcium-mediated virus reactivation in KSHV-infected dermal microvascular endothelial cells was blocked by cyclosporine. Furthermore, retroviral transduction with plasmid DNA encoding VIVIT, a peptide specifically blocking calcineurin-NFAT interactions, inhibited calcium-dependent KSHV reactivation. By contrast, chemical induction of lytic-phase infection by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate was blocked by protein kinase C inhibitors, but not by calcineurin inhibitors. In summary, calcineurin-dependent signal transduction, an important signaling cascade in vivo, induces calcium-dependent KSHV replication, providing a possible target for the design of antiherpesvirus strategies in KSHV-infected patients.

Blood-brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis.

The blood-brain barrier (BBB) plays a critical role in regulating cell trafficking through the central nervous system (CNS) due to several unique anatomical features, including the presence of interendothelial tight junctions that form impermeable seals between the cells. Previous studies have demonstrated BBB perturbations during human immunodeficiency virus encephalitis (HIVE); however, the basis of these permeability changes and its relationship to infiltration of human immunodeficiency virus type 1 (HIV-1)-infected monocytes, a critical event in the pathogenesis of the disease, remains unclear. In this study, we examined CNS tissue from HIV-1-seronegative patients and HIV-1-infected patients, both with and without encephalitis, for alterations in BBB integrity via immunohistochemical analysis of the tight junction membrane proteins, occludin and zonula occludens-1 (ZO-1). Significant tight junction disruption (P < 0.001), as demonstrated by fragmentation or absence of immunoreactivity for occludin and ZO-1, was observed within vessels from subcortical white matter, basal ganglia, and, to a lesser extent, cortical gray matter in patients who died with HIVE. These alterations were also associated with accumulation of activated, HIV-1-infected brain macrophages, fibrinogen leakage, and marked astrocytosis. In contrast, no significant changes (P > 0.05) were observed in cerebellar tissue from patients with HIVE compared to HIV-seronegative patients or HIV-1-infected patients without encephalitis. Our findings demonstrate that tight junction disruption is a key feature of HIVE that occurs in regions of histopathological alterations in association with perivascular accumulation of activated HIV-1-infected macrophages, serum protein extravasation, and marked astrocytosis. We propose that disruption of this key BBB structure serves as the main route of HIV-1-infected monocyte entry into the CNS.

Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8.

Human herpesvirus 8 (HHV8) infects Kaposi's sarcoma (KS) spindle cells in situ, as well as the lesional endothelial cells considered to be spindle cell precursors. The HHV8 genome contains several oncogenes, suggesting that infection of endothelial and spindle cells could induce cellular transformation and tumorigenesis and promote the formation of KS lesions. To investigate the potential of HHV8 infection of endothelial cells to contribute to the development of KS, we have developed an in vitro model utilizing dermal microvascular endothelial cells that support significant HHV8 infection. In contrast to existing in vitro systems used to study HHV8 pathogenesis, the majority of dermal endothelial cells are infected with HHV8 and the viral genome is maintained indefinitely. Infection is predominantly latent, with a small percentage of cells supporting lytic replication, and latency is responsive to lytic induction stimuli. Infected endothelial cells develop a spindle shape resembling that of KS lesional cells and show characteristics of a transformed phenotype, including loss of contact inhibition and acquisition of anchorage-independent growth. These results describe a relevant model system in which to study virus-host interactions in vitro and demonstrate the ability of HHV8 to induce phenotypic changes in infected endothelial cells that resemble characteristics of KS spindle cells in vivo. Thus, our results are consistent with a direct role for HHV8 in the pathogenesis of KS.

HIV-1 induction of CD40 on endothelial cells promotes the outgrowth of AIDS-associated B-cell lymphomas.

Human immunodeficiency virus (HIV)-1 infection is associated with the development of aggressive extranodal B-cell non-Hodgkin's lymphomas. Using microvascular endothelial cell (MVEC)-enriched bone marrow stromal cultures, HIV infection of stromal MVECs from lymphoma patients induced the outgrowth of malignant B cells. MVECs were the only HIV-infected cells in the stroma, and purified brain MVECs also induced a phenotype supportive of neoplastic B-cell attachment and proliferation. HIV infection of MVECs stimulated surface expression of CD40 and allowed preferential induction of the vascular cell adhesion molecule VCAM-1 after CD40 triggering. B-lymphoma cells expressed the CD40 ligand (CD40L), and blocking of CD40-CD40L interactions between HIV-infected MVECs and B-lymphoma cells inhibited B-cell attachment and proliferation. These observations suggest that HIV promotes B-lymphoma cell growth through facilitating attachment of lymphoma cells to HIV-infected MVECs and represent a novel mechanism through which viruses may induce malignancies.

Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells.

The cytoadherence of Plasmodium falciparum-infected erythrocytes was studied using immortalized human brain capillary endothelial cells. The immortalized cells, denoted as BB19, derived from the human brain endothelium, were transformed with the E6E7 genes of human papilloma virus and retained their endothelial nature, i.e. tubule formation occurred with Matrigel as a substratum and the cells stained positive for Factor VIII-related antigen, or vonWillebrand's factor. Surface expression of ICAM-1, VCAM, E-selectin, and CD36 was demonstrated by immunofluorescence staining with monoclonal antibodies to these ligands. Exposure to cytokines (TNF, IFN gamma, IL-1 alpha, and IL-6) and lipopolysaccharide resulted in an increase in expression of ICAM-1, VCAM, E-selectin, and CD36. The BB19 cells bound P. falciparum-infected red blood cells with both the FCR-3 and the ITO4 strains. Antibodies to CD36 and ICAM-1 partially inhibited the binding of the FCR-3 and the ITO4 lines, respectively. These findings suggest that BB19 cells may be useful in the analysis of receptor-based cytoadherence and sequestration, as well as in the cell biology of microvessel formation.

Sequences regulating tropism of human immunodeficiency virus type 1 for brain capillary endothelial cells map to a unique region on the viral genome.

Two infectious molecular clones of human immunodeficiency virus type 1, NL4-3 and JR-CSF, differ in their abilities to productively infect human brain capillary endothelial (HBCE) cells. The phenotypes of recombinants between these two molecular strains were examined to identify viral sequences responsible for the difference in HBCE cell tropism between the two parental strains. Our results indicate that HBCE cell tropism maps to a region that encompasses the C1 region of env and includes overlapping reading frames for the accessory genes vpr, vpu, tat, and rev. This region was unique for HBCE cell tropism and did not cosegregate with either macrophage or T-cell line tropism. However, several recombinant clones displayed dual tropism for both HBCE cells and macrophages. These endothelial cell- and macrophage-tropic strains may have a unique pathogenic advantage by entering the brain via HBCE cells and subsequently infecting microglial cells with high efficiency, leading to the induction of human immunodeficiency virus dementia.

Human immunodeficiency virus infection of bone marrow endothelium reduces induction of stromal hematopoietic growth factors.

The majority of human immunodeficiency virus (HIV)-seropositive patients develop bone marrow abnormalities associated with hematopoietic malfunction during the progression of disease. One important manifestation of HIV-associated hematopoietic dysfunction is that after myelosuppression, bone marrow recovery, a process known to be mediated in part by the production of stromal cell-derived hematopoietic growth factors, is impaired. We sought to test the hypothesis that bone marrow stromal cells are infected by HIV-1 in vivo and that production of certain stromal cell-derived hematopoietic growth factors is deficient as a consequence. In this report, we demonstrate that bone marrow microvascular endothelial cells (MVEC), a key element of the stroma, are the predominant cells infected by HIV (5% to 20%) in bone marrow stromal cultures obtained from 11 consecutive HIV-seropositive patients. Although HIV-infected stromal cultures enriched for MVEC constitutively express normal levels of interleukin (IL)-4, IL-6, granulocyte (G)-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and Steel factor, IL-1 alpha-induced release of IL-6 and G-CSF is significantly reduced in these cultures. These observations suggest that HIV infection of bone marrow MVEC reduces the capacity of hematopoietic stroma to respond to regulatory signals that normally augment blood cell production during periods of increased demand.

Growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages.

Stimulation of monocytes/macrophages with activated nonadherent cells allows productive nonlytic growth of human cytomegalovirus (HCMV), but the viral replication cycle is delayed relative to replication of HCMV in human fibroblasts. Analysis of infected monocyte-derived macrophage (MDM) mRNA for major immediate-early (MIE 86, 72, and 55) and late (pp65 and gB) gene expression by reverse transcription PCR indicates that transcription peaks at 3 and 7 days postinfection (dpi), respectively. In contrast, in human fibroblast controls, mRNA for MIE and late gene expression peaked at 5 and 48 h postinfection, respectively. Consistent with reverse transcription PCR experiments, double-label antibody experiments first detected MIE antigen expression at 12 h postinfection, peaking at 3 dpi, and late (pp65 or gB) antigen expression at 5 dpi, peaking at 7 dpi. MIE antigen was not detected between 3 and 7 dpi but reappeared and was coexpressed with pp65 in enlarged MDM nuclei at 7 dpi. After 7 dpi, macrophages with numerous vacuoles containing large amounts of pp65 and gB were observed in culture. These vacuoles were frequently seen at cellular contact points, suggesting that cell-to-cell transfer of virus was the major mode of viral transmission. Consistent with this observation, infectious virus was recovered from MDM cellular lysates but not culture supernatant. The delayed growth and compartmentalization of HCMV in macrophages may allow the cell to accommodate the viral replication cycle without cell lysis. In addition, the macrophage may function as a vehicle for cell-to-cell transmission of HCMV.

Differential role of long terminal repeat control elements for the regulation of basal and Tat-mediated transcription of the human immunodeficiency virus in stimulated and unstimulated primary human macrophages.

Primary human macrophages induced to differentiate through contact with autologous activated nonadherent cells were used to investigate the transcriptional mechanisms involved in reactivation of human immunodeficiency virus (HIV) replication. Through transient transfection experiments with an HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase reporter construct, we show that macrophage differentiation results in a 20-fold upregulation of basal LTR activity. To identify sequence elements responsive to the differentiation process, point mutations introduced into the LTR were tested in differentiated and undifferentiated macrophages. Several elements were identified as positive regulators of basal transcription. TATA, Sp1, and NF-kappa B binding sites were the most influential. The low-affinity site for LBP-1 (UBP-1) functioned as a negative regulator of LTR activity in undifferentiated macrophages, but this influence was lost upon differentiation. When tat was cotransfected into the expression system, the requirement for LTR elements identified as important for positive regulation of basal transcription remained in undifferentiated macrophages. Interestingly, however, the mutations in positive control elements which debilitated activity in undifferentiated macrophages had no effect on LTR activity in differentiated macrophages. Thus, it appears that while HIV-LTR activity is highly dependent on cellular transcription factors in undifferentiated cells, in differentiated macrophages the viral protein Tat confers pliability on the LTR and facilitates autonomy from absolute cellular control mechanisms. In vivo, release from either positive or negative regulation via cellular proteins may facilitate reactivation of HIV in macrophages.

HIV infection of human brain capillary endothelial cells--implications for AIDS dementia.

We have demonstrated that human brain capillary endothelial (HBCE) cells, unlike umbilical or aortic endothelial cells are permissively infected by HIV. HIV infection of HBCE cells is noncytolytic and is mediated by a CD4- and GalCer-independent mechanism, implying that HBCE cell tropic strains utilize a unique receptor. The V3 loop of gp120 appears to be important in this reaction. T-cell tropic but not brain-derived macrophage tropic HIV strains selectively infect brain endothelium suggesting that T-cell tropism is important for HIV entry through the blood-brain barrier (BBB). The ability of HIV to infect cells that compose the BBB implies that the virus may be directly involved in the BBB dysfunction observed in AIDS patients. HIV infection of HBCE cells may allow the flow of cytokines or toxic metabolites from the circulating blood into the brain parenchyma either by disrupting tight junctions or by altering the ability of the cells to regulate transport of substances across the BBB by transcytosis. HIV infection may also result in endothelial cell-induced astrocytosis by release of cytotoxic substances or modulation of abluminal surface antigens which contact astrocytic foot processes. Finally, HIV infection of the brain endothelium could facilitate virus entry to the CNS either by infection of HBCE cells or via entry of HIV-infected leucocytes. The establishment of our in vitro HIV-HBCE cell system will allow us to explore the potential mechanisms which mediate AIDS dementia.

Human immunodeficiency virus infection of human brain capillary endothelial cells occurs via a CD4/galactosylceramide-independent mechanism.

Neuropathologic studies of AIDS patients have shown that brain capillary endothelial cells are a cellular target for human immunodeficiency virus (HIV) in vivo. We have established in vitro cultures of primary human brain capillary endothelial (HBCE) cells. Using this model system, we have shown a significant HIV infection of HBCE cells that is productive yet noncytopathic. The infection is mediated by a cellular interaction with gp120 that does not involve CD4 or galactosylceramide. HIV infection of HBCE cells may contribute to AIDS-associated neuropathology by disturbing the physiology of the endothelium and directly or indirectly facilitating dissemination of virus to the central nervous system.

The effect of human cytomegalovirus on the production and biologic action of interleukin-1.

The effect of mycoplasma-free human cytomegalovirus (HCMV) on the production and biologic activity of interleukin-1 (IL-1) from peripheral blood monocytes was examined. The use of biologic thymocyte assays revealed a time-dependent decrease in the IL-1 activity of both HCMV-challenged and control monocytes after initiation of culture. A decrease in the amount of IL-1 beta secreted as measured by ELISA was also detected. The amount of IL-1 beta secreted by HCMV-challenged cells was always greater than that produced by control cultures at similar times. Despite containing higher levels of IL-1 beta, supernatants from challenged cells were markedly less effective in supporting thymocyte proliferation. It is proposed that this is due to the concomitant production of an inhibitor of IL-1 activity from HCMV-challenged monocyte cultures.