Endothelial injury is a major manifestation of septic shock induced by LPS. Recently, LPS was shown to induce apoptosis in different types of endothelial cells. In this study, we observed that pretreatment with vascular endothelial growth factor (VEGF), a known cell survival factor, blocked LPS-induced apoptosis in endothelial cells. We then further defined this LPS-induced apoptotic pathway and its inhibition by VEGF. We found that LPS treatment increased caspase-3 and caspase-1 activities and induced the cleavage of focal adhesion kinase. LPS also augmented expression of the pro-apoptotic protein Bax and the tumor suppressor gene p53. The pro-apoptotic Bax was found to translocate to the mitochondria from the cytosol following stimulation with LPS. Pretreatment of endothelial cells with VEGF inhibited the induction of both Bax and p53 as well as the activation of caspase-3. These data suggest that VEGF inhibits LPS-induced endothelial apoptosis by blocking pathways that lead to caspase activation.

The integrity of the blood-brain barrier (BBB) is critical for normal brain function. Neuropathological abnormalities in AIDS patients have been associated with perivascular HIV-infected macrophages, gliosis, and abnormalities in the permeability of the BBB. The processes by which HIV causes these pathological conditions are not well understood. To characterize the mechanism by which HIV-1 Tat protein modulates human brain microvascular endothelial cell (HBMEC) functions, we studied the effects of HIV-1 Tat in modulating HBMEC apoptosis and permeability. Treatment of HBMEC with HIV-1 Tat led to Flk-1/KDR and Flt-4 receptor activation and the release of NO. The protein levels of endothelial NO synthase (NOS) and inducible NOS were increased by HIV-1 Tat stimulation. Importantly, HIV-1 Tat caused apoptosis of HBMEC, as evidenced by changes in the cleavage of poly(A)DP-ribose polymerase, DNA laddering, and incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay). HIV-1 Tat-mediated apoptosis in HBMEC was significantly inhibited in the presence of N-nitro-l-arginine methyl ester (an inhibitor of NOS) and wortmannin (a phosphoinositol 3-kinase inhibitor). Furthermore, HIV-1 Tat treatment significantly increased HBMEC permeability, and pretreatment with both N-nitro-l-arginine methyl ester and wortmannin inhibited the Tat-induced permeability. Taken together, these results indicate that dysregulated production of NO by HIV-1 Tat plays a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB.

HIV-1 Tat, in addition to its critical role in viral transcription, is secreted from infected cells and can act as a proto-cytokine. We studied the effects of HIV-1 Tat in primary human microvascular endothelial cells of lung origin and found that it caused apoptosis. This apoptosis occurred without induction of either Fas or TNF, known mediators of programmed cell death. Tat, like Fas ligand, induced cleavage of chromatin structure, as evidenced by changes in DNA laddering, incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay), and mono- or oligonucleosomes. Furthermore, Tat treatment caused cleavage of poly(A/DP)-ribose polymerase, a substrate of caspases. Caspase-3, but not caspase-9, was activated following treatment of primary human microvascular endothelial cells of lung origin with either Tat or anti-Fas agonist Ab (anti-Fas). Inhibition of caspase-3 activity markedly reduced apoptosis. Although Fas-mediated apoptosis involved changes in Bcl-2, Bax, and Bad regulatory proteins, such alterations were not observed with Tat. Taken together, these data demonstrate that HIV-1 Tat is able to activate apoptosis in microvascular endothelium by a mechanism distinct from TNF secretion or the Fas pathway.

No abstract

Nef is a viral regulatory protein of the human immunodeficiency virus (HIV) that has been shown to contribute to disease progression. Among its putative effects on T cell functions are the down-regulation of CD4 and major histocompatibility class I surface molecules. These effects occur in part via Nef interactions with intracellular signaling molecules. We sought to better characterize the effects of HIV Nef on T cell function by examining chemotaxis in response to stromal cell-derived factor-1␣ (SDF-1␣) as well as CXCR4 signaling molecules. Here, we report the novel observation that HIV Nef inhibited chemotaxis in response to SDF-1␣ in both Jurkat T cells and primary peripheral CD4؉ T lymphocytes. Our data indicate that HIV Nef altered critical downstream molecules in the CXCR4 pathway, including focal adhesion kinases. These findings suggest that HIV Nef may blunt the T cell response to chemokines.

SUMMARY HIV-1 Nef plays important roles in HIV-1 replication and pathogenesis. It is translated from completely spliced HIV-1 RNA, its expression is inherently regulated at the levels of viral DNA transcription and RNA splicing. Here we show that Sam68 cytoplasmic mutants potently suppress Nef expression. The suppression requires Sam68 domain aa269-321 and is correlated with its ability to induce stress granules. In addition, the suppression is specific to Nef, and direct binding to nef mRNA 3′UTR confers the suppression specificity. Furthermore, nef mRNA is targeted to and enriched in these induced stress granules. Importantly, Nef suppression occurs in the context of HIV-1 infection of CD4+ T lymphocytes with little MHC I and CD4 down-regulation. Taken together, these results demonstrate that stress granule induction and nef mRNA sequestration account for this translational suppression of Nef expression and offers a new strategy for development of anti-HIV therapeutics to buttress our fight against HIV/AIDS.

HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5∼2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay.

The stromal cell-derived factor-1 (SDF-1) is an alpha chemokine that binds to the CXCR4 receptor. Knock-out studies in mice demonstrate that this ligand-receptor pair is essential in hematopoiesis. One function of SDF-1 appears to be the regulation of migration of hematopoietic progenitor cells. We previously characterized signal transduction pathways induced by SDF-1 in human hematopoietic progenitors and found tyrosine phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK), the adaptor molecule p130 Cas, and the cytoskeletal protein paxillin. To better understand the functional role of signaling molecules connecting the CXCR4 receptor to the process of hematopoietic migration, we studied SDF-1–mediated pathways in a model hematopoietic progenitor cell line (CTS), as well as in primary human bone marrow CD34+cells. We observed that several other focal adhesion components, including focal adhesion kinase (FAK) and the adaptor molecules Crk and Crk-L, are phosphorylated on SDF-1 stimulation. Using a series of specific small molecule inhibitors, both protein kinase C (PKC) and phosphoinositide-3 kinase (PI-3K) appeared to be required for SDF-1–mediated phosphorylation of focal adhesion proteins and the migration of both CTS and primary marrow CD34+ cells, whereas the mitogen-activated protein kinases ERK-1 and -2 were not. These studies further delineate the molecular pathways mediating hematopoietic progenitor migration and response to an essential chemokine, SDF-1.