Kaposi sarcoma herpesvirus K5 removes CD31/PECAM from endothelial cells

The transmembrane ubiquitin ligase K5/MIR2 of Kaposi sarcoma herpesvirus (KSHV) mediates internalization and lysosomal degradation of glycoproteins involved in antigen presentation and co-stimulation. In endothelial cells (ECs), K5 additionally reduced expression of CD31/platelet-endothelial cell adhesion molecule (PECAM), an adhesion molecule regulating cell-cell interactions of ECs, platelets, monocytes, and T cells. K5 also reduced EC migration, a CD31-dependent process. Unlike other K5 substrates, both newly synthesized and pre-existing CD31 molecules were targeted by K5. K5 was transported to the cell surface and ubiquitinated pre-existing CD31, resulting in endocytosis and lysosomal degradation. In the endoplasmic reticulum, newly synthesized CD31 was degraded by proteasomes, which required binding of phosphofurin acidic cluster sorting protein-2 (PACS-2) to acidic residues in the carboxyterminal tail of K5. Thus, CD31, a novel target of K5, is efficiently removed from ECs by a dual degradation mechanism that is regulated by the subcellular sorting of the ubiquitin ligase. K5-mediated degradation of CD31 is likely to affect EC function in KS tumors.     

AIDS, Kaposi sarcoma, and the lymphatic system: update and reflections

Based on ongoing basic and clinical investigations, further evidence is presented that the AIDS-Kaposi sarcoma (AIDS-KS) complex involves a progressive disturbance of the blood-lymph circulatory loop of fluid, macromolecules, and migrating cells. We first surveyed the spectrum of vascular abnormalities including Kaposi sarcoma (KS) found in the AIDS/ARC population, then non-invasively imaged lymphatic system abnormalities in AIDS-KS by whole body lymphangioscintigraphy, and finally examined the biologic behavior of AIDS-KS cells in long-term tissue culture. These observations are viewed in terms of the lymphatic and blood vascular route of entry and transport of free and cell-associated virus and other opportunistic pathogens as well as poorly understood host endothelial-immune system interactions.     

The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned medium

The CC chemokine receptor 8 (CCR8) is expressed on monocytes andtype 2 T lymphocytes. CCR8 is the sole receptor for the human CCchemokine I-309, as well as for viral monocyte inflammatory protein-I(vMIP-I), a human chemokine homologue induced in human cells by theKaposi sarcoma-related human herpesvirus-8. Recently it was found thatI-309 messenger RNA and protein are expressed by human umbilical veinendothelial cells (HUVECs) and that the secretion of endothelial I-309is stimulated by apolipoprotein(a). I-309, vMIP-I, and the conditionedmedium from apolipoprotein(a)-stimulated HUVECs induce endothelialchemotaxis. A polyclonal anti-CCR8 antibody and a newly developedmurine monoclonal antibody against CCR8 inhibited this activity. TheG-protein inhibitor pertussis toxin also inhibited endothelialchemotaxis, providing further evidence for a chemokinereceptor-mediated effect. Endothelial cells contain CCR8 mRNA as shownby RNA blot analysis as well by direct sequence analysis.Immunohistochemical studies identified CCR8 and I-309 on theendothelium of human atherosclerotic plaques and in endothelial-derived spindle cells of Kaposi sarcoma. These results indicate that CCR8 is anendothelial receptor that may modulate endothelial function.     

Mechanotransduction in lymphatic endothelial cells

Initial lymphatic vessel endothelial cells are connected to the surrounding elastic fibers by fibrillin anchoring filaments that have been hypothesized to favor interstitial fluid drainage in edema pulling apart interendothelial junctions. We hypothesized a biochemical mechanism involving mechanotransduction. This study was designed to verify whether a relation exists between focal adhesion molecules and anchoring filaments and whether they may transduce extracellular forces to the nucleus. We first performed an immunohistochemical study on human skin cryostat sections to evaluate whether fibrillin and alphav-beta3 integrins, FAK and fibrillin, or alphav-beta3 integrins and FAK co-localize in lymphatic endothelium. We observed that integrins and FAK co-localize and that fibrillin filament attachment sites to endothelial cells merge with these molecules. These data may suggest that fibrillin anchoring filaments are connected to endothelial cells through focal adhesions. Mechanotransduction was investigated applying static stretching to bovine thoracic duct segments and lymphatic endothelial cells cultured on elastic membranes and immunohistochemically evaluating the expression of ERK1/2. Under stretching conditions, ERK1/2 labels the nucleus. Western blotting on cultured cells confirmed the presence of ERK1/2 in stretched cells. Based on our data we speculate that anchoring filaments may trigger a focal adhesion-mediated cascade of mechanotransduction toward the nucleus for genetic modulation and thus contribute to endothelial adaptation to interstitial requirements.     

HIV fragments detected in Kaposi sarcoma tumor cells in HIV-infected patients

Kaposi sarcoma (KS) is a malignant vascular neoplasm caused by KS-associated herpesvirus (KSHV) infection. HIV plays a major role in KS pathogenesis. KS in HIV usually produces more malignant features than classic KS. Despite the close KS–HIV relationship, no study has reported the existence of HIV in KS tissue. We used ddPCR to detect HIV and KSHV in HIV⁺ KS samples and classic KS control. We verified KS cell types through immunohistochemistry and applied hypersensitive in situ hybridization (ISH) to detect HIV and KSHV in tumor cells. Furthermore, we co-stained samples with ISH and immunohistochemistry to identify HIV and KSHV in specific cell types. Regarding pathological stages, the KS were nodular (58.3%), plaque (33.3%), and patch (8.3%) tumors. Moreover, ddPCR revealed HIV in 58.3% of the KS samples. ISH revealed positive Pol/Gag mRNA signals in CD34 ⁺ tumor cells from HIV ⁺ patients (95.8%). HIV signals were absent in macrophages and other inflammatory cells. Most HIV ⁺ KS cells showed scattered reactive particles of HIV and KSHV. We demonstrated that HIV could infect CD34 ⁺ tumor cells and coexist with KSHV in KS, constituting a novel finding. We hypothesized that the direct KSHV–HIV interaction at the cellular level contributes to KS oncogenesis.     

Prognostic value of quantitative Kaposi sarcoma--associated herpesvirus load in posttransplantation Kaposi sarcoma

Reactivation of human beta-herpesviruses (cytomegalovirus [CMV], human herpesvirus [HHV]-6, and HHV-7) in nonimmunocompromised hosts is rare. Because these viruses are susceptible to reactivation by cytokines and stress-related mechanisms, the incidence of their reactivation was investigated among 120 patients during stress related to critical illness and compared with findings among 50 healthy volunteers. Human beta-herpesvirus DNA was found in 65% of critically ill patients (60% men; mean age, 63 years) who required admission to an intensive care unit for medical (40%) or surgical (53%) indications or trauma (7%). HHV-6 reactivation was higher in critically ill patients than in healthy volunteers (54/101 vs. 0/50; P=.001). All patients except 1 were confirmed as HHV-6 variant A (mean virus load, 5066 copies/10(6) peripheral blood leukocytes). The reactivation of HHV-6A did not affect disease severity and outcome. No significant reactivation of HHV-7 or CMV was demonstrated among the critically ill patients. These findings contribute to the less-defined epidemiology of HHV-6A infection.     

Kaposi sarcoma-associated herpesvirus (KSHV) induces heme oxygenase-1 expression and activity in KSHV-infected endothelial cells

Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)      

Vascular endothelial growth factor-C enhances radiosensitivity of lymphatic endothelial cells

Radiation therapy after lymph node dissection increases the risk of developing painful and incurable lymphedema in breast cancer patients. Lymphedema occurs when lymphatic vessels become unable to maintain proper fluid balance. The sensitivity of lymphatic endothelial cells (LECs) to ionizing radiation has not been reported to date. Here, the radiosensitivity of LECs in vitro has been determined using clonogenic survival assays. The ability of various growth factors to alter LEC radiosensitivity was also examined. Vascular endothelial growth factor (VEGF)-C enhanced radiosensitivity when LECs were treated prior to radiation. VEGF-C-treated LECs exhibited higher levels of entry into the cell cycle at the time of radiation, with a greater number of cells in the S and G2/M phases. These LECs showed higher levels of γH2A.X—an indicator of DNA damage—after radiation. VEGF-C did not increase cell death as a result of radiation. Instead, it increased the relative number of quiescent LECs. These data suggest that abundant VEGF-C or lymphangiogenesis may predispose patients to radiation-induced lymphedema by impairing lymphatic vessel repair through induction of LEC quiescence.     

Autonomous phagosomal degradation and antigen presentation in dendritic cells

Phagocytosis plays a critical role in both innate and adaptive immunity. Phagosomal fusion with late endosomes and lysosomes enhances proteolysis, causing degradation of the phagocytic content. Increased degradation participates in both innate protection against pathogens and the production of antigenic peptides for presentation to T lymphocytes during adaptive immune responses. Specific ligands present in the phagosomal cargo influence the rate of phagosome fusion with lysosomes, thereby modulating both antigen degradation and presentation. Using a combination of cell sorting techniques and single phagosome flow cytometry-based analysis, we found that opsonization with IgG accelerates antigen degradation within individual IgG-containing phagosomes, but not in other phagosomes present in the same cell and devoid of IgG. Likewise, IgG opsonization enhances antigen presentation to CD4(+) T lymphocytes only when antigen and IgG are present within the same phagosome, whereas cells containing phagosomes with either antigen or IgG alone failed to present antigen efficiently. Therefore, individual phagosomes behave autonomously, in terms of both cargo degradation and antigen presentation to CD4(+) T cells. Phagosomal autonomy could serve as a basis for the intracellular discrimination between self and nonself antigens, resulting in the preferential presentation of peptides derived from opsonized, nonself antigens.     

p21Cip1 levels differentially regulate turnover of mature endothelial cells, endothelial progenitor cells, and in vivo neovascularization

p21(Cip1) (p21) controls cell cycle progression and apoptosis in mature endothelial cells (ECs) and regulates size and cycling of the hematopoietic progenitor cell pool. Because circulating endothelial progenitor cells (EPCs) contribute to postnatal neovascularization in addition to mature ECs, we investigated the regulation of ECs and EPCs in p21-deficient mice. Mature aortic EC proliferation was increased in homozygous p21(-/-) and heterozygous p21(+/-) mice, in which p21 protein levels are reduced to one third of wild-type (WT). In contrast, apoptosis sensitivity was increased by 3.5-fold only in p21(-/-), but not in p21(+/-) mice. Consistently, in vivo apoptosis of ECs within areas of neovascularization was elevated in p21(-/-) but not in p21(+/-) mice. EPC numbers were elevated 2-fold in p21(-/-) mice compared with WT (P<0.001), and clonal expansion capacity of EPCs was increased from 25+/-4 (WT) to 57+/-8 colony-forming units in p21(-/-) mice (P<0.005). EPC numbers and expansion were likewise increased in p21(+/-) mice. As the integrative endpoint, in vivo neovascularization reflecting all p21-affected parameters was increased over WT only in p21(+/-) (P<0.001), but not in p21(-/-) mice. In conclusion, reduced p21 protein levels of mice lacking one p21 allele are associated with increased proliferation of ECs and EPCs, whereas survival of ECs to apoptotic stimuli in vitro and in vivo is not impaired. Under these conditions, neovascularization was increased. In contrast, complete p21 deficiency did not result in an increased neovascularization despite increased mature EC and EPC proliferation. This may be due to the sensitization of ECs against apoptosis.     

Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells and B cells across hepatic sinusoidal endothelium

BackgroundAdult inflammatory liver diseases are driven by lymphocyte infiltration of liver tissue. Lymphocyte recruitment occurs within the unique low shear environment of the hepatic sinusoids. These channels are lined by hepatic sinusoidal endothelial cells (HSEC) which lack conventional adhesion receptors such as selectins, leading us to investigate the role of unconventional adhesion molecules. The common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) is a C-type lectin that has been shown to mediate lymphocyte recruitment to lymphatic endothelium.MethodsWe studied the expression of CLEVER-1 in human liver tissue by immunohistochemistry and immunofluorescence. We proceeded to confirm the expression of CLEVER-1 in isolated HSEC in vitro. Flow based adhesion assays were performed with adhesion blocking antibodies to elucidate the functional role of CLEVER-1. These studies were extended to visualise lymphocyte recruitment and transendothelial migration with immunofluorescence staining and confocal microscopy. Migration of lymphocyte subsets was also studied with tracking software.FindingsCLEVER-1 was expressed at sites of lymphocyte trafficking within the inflamed human liver, particularly the hepatic sinusoids, neovessels of the fibrous septum, and tertiary lymphoid follicles. Furthermore, CLEVER-1 was also detected in the sinusoids and supplying vessels of hepatocellular carcinomas. Functional assays with HSEC showed that CLEVER-1 mediated the transendothelial migration of peripheral blood lymphocytes with preferential activity for regulatory T cells and B cell subsets. Detailed confocal analysis visualised a novel transcellular route of migration by regulatory T cells. B cells also demonstrated altered migratory capacity compared with T cells.InterpretationThese results suggest that CLEVER-1 has a role in modulating the recruitment of regulatory T cells and B cells to the human liver. This C-type lectin could be a potential therapeutic target for chronic inflammatory liver disease and hepatocellular cancer.FundingWellcome Trust.     

Acetoacetate and beta-hydroxybutyrate differentially regulate endothelin-1 and vascular endothelial growth factor in mouse brain microvascular endothelial cells.

Insulin-dependent diabetes mellitus (IDDM), is characterized by a lack of insulin production from beta cells in the pancreas. One of the metabolic consequences of this insulin deficit is an increased hepatic synthesis of ketone bodies, resulting in a serious medical complication, diabetic ketoacidosis (DKA). DKA, in turn, has been associated with the development of cerebral edema. The severity of this complication ranges from death to a subclinical presentation, but seems to be invariably present to some degree. The etiology of the cerebral edema is unknown, but changes in osmolality, pH, and insulin effects on the blood-brain barrier have all been suggested as possible culprits. Blood-brain barrier impermeability is maintained by the endothelial cells (EC) lining the blood vessels. Thus, it would seem likely that alterations in EC function would be necessary for the development of cerebral edema. However, no studies have examined the effects of ketone bodies on brain endothelial cells. The two major ketone bodies in DKA are acetoacetate (AcAc) and beta-hydroxybutyrate (BOHB). In the present study we examined the effect of these ketone bodies on a major intracellular signalling pathway. The changes in intracellular calcium concentration, and the production of two vasoactive peptides, endothelin-1 (ET-1) and vascular permeability factor (VPF/VEGF) in mouse brain microvascular endothelial cells (MBMEC). The present studies demonstrate the BOHB can increase vascular permeability factor. In contrast, AcAc increases the production of the potent vasoconstrictor, endothelin-1. This data would suggest that brain ECs are potential targets of the metabolic alterations in DKA.     

G-protein-coupled receptor S1P1 acts within endothelial cells to regulate vascular maturation

Sphingosine-1-phosphate (S1P) stimulates signaling pathways via G-protein-coupled receptors and triggers diverse cellular processes, including growth, survival, and migration. In S1P1 receptor-deficient embryos, blood vessels were incompletely covered by vascular smooth muscle cells (VSMCs), indicating the S1P1 receptor regulates vascular maturation. Because S1P1 receptor expression is not restricted to a particular cell type, it was not known whether the S1P1 receptor controlled VSMC coverage of vessels in a cell-autonomous fashion by functioning directly in VSMCs or indirectly through its activity in endothelial cells (ECs). By using the Cre/loxP system, we disrupted the S1P1 gene solely in ECs. The phenotype of the conditional mutant embryos mimicked the one obtained in the embryos globally deficient in S1P1. Thus, vessel coverage by VSMCs is directed by the activity of the S1P1 receptor in ECs.