Genital tract sequestration of SIV following acute infection.

We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus "spillover" into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves.

Systems serology-based comparison of antibody effector functions induced by adjuvanted vaccines to guide vaccine design.

The mechanisms by which antibodies confer protection vary across vaccines, ranging from simple neutralization to functions requiring innate immune recruitment via Fc-dependent mechanisms. The role of adjuvants in shaping the maturation of antibody-effector functions remains under investigated. Using systems serology, we compared adjuvants in licensed vaccines (AS01B/AS01E/AS03/AS04/Alum) combined with a model antigen. Antigen-naive adults received two adjuvanted immunizations followed by late revaccination with fractional-dosed non-adjuvanted antigen ( NCT00805389 ). A dichotomy in response quantities/qualities emerged post-dose 2 between AS01B/AS01E/AS03 and AS04/Alum, based on four features related to immunoglobulin titers or Fc-effector functions. AS01B/E and AS03 induced similar robust responses that were boosted upon revaccination, suggesting that memory B-cell programming by the adjuvanted vaccinations dictated responses post non-adjuvanted boost. AS04 and Alum induced weaker responses, that were dissimilar with enhanced functionalities for AS04. Distinct adjuvant classes can be leveraged to tune antibody-effector functions, where selective vaccine formulation using adjuvants with different immunological properties may direct antigen-specific antibody functions.

The TRIM5 gene modulates penile mucosal acquisition of simian immunodeficiency virus in rhesus monkeys.

There is considerable variability in host susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, but the host genetic determinants of that variability are not well understood. In addition to serving as a block for cross-species retroviral infection, TRIM5 was recently shown to play a central role in limiting primate immunodeficiency virus replication. We hypothesized that TRIM5 may also contribute to susceptibility to mucosal acquisition of simian immunodeficiency virus (SIV) in rhesus monkeys. We explored this hypothesis by establishing 3 cohorts of Indian-origin rhesus monkeys with different TRIM5 genotypes: homozygous restrictive, heterozygous permissive, and homozygous permissive. We then evaluated the effect of TRIM5 genotype on the penile transmission of SIVsmE660. We observed a significant effect of TRIM5 genotype on mucosal SIVsmE660 acquisition in that no SIV transmission occurred in monkeys with only restrictive TRIM5 alleles. In contrast, systemic SIV infections were initiated after preputial pocket exposures in monkeys that had at least one permissive TRIM5 allele. These data demonstrate that host genetic factors can play a critical role in restricting mucosal transmission of a primate immunodeficiency virus. In addition, we used our understanding of TRIM5 to establish a novel nonhuman primate penile transmission model for AIDS mucosal pathogenesis and vaccine research.

Polysaccharide and conjugate vaccines to Streptococcus pneumoniae generate distinct humoral responses.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia, bacteremia, and meningitis in older adults worldwide. Two pneumococcal vaccines containing S. pneumoniae capsular polysaccharides are in current use: the polysaccharide vaccine PPSV23 and the glycoconjugate vaccine PCV13. In clinical trials, both vaccines elicit similar opsonophagocytic killing activity. In contrast to polysaccharide vaccines, conjugate vaccines have shown consistent efficacy against nasopharyngeal carriage and noninvasive pneumonia overall and for some prevalent individual serotypes. Given these different clinical profiles, it is crucial to understand the differential immunological responses induced by these two vaccines. Here, we used a high-throughput systems serology approach to profile the biophysical and functional features of serum antibodies induced by PCV13 and PPSV23 at 1 month and 1 year. In comparison with PPSV23, PCV13 induced higher titers across antibody isotypes; more durable antibody responses across immunoglobulin G (IgG), IgA, and IgM isotypes; and increased antigenic breadth. Although titers measured in opsonophagocytic activity (OPA) assays were similar between the two groups, confirming what was observed in clinical studies, serum samples from PCV13 vaccinees could induce additional non-OPA antibody-dependent functions, including monocyte phagocytosis and natural killer cell activation. In a multivariate modeling approach, distinct humoral profiles were demonstrated in each arm. Together, these results demonstrate that the glycoconjugate PCV13 vaccine induces an antigenically broader, more durable, polyfunctional antibody response. These findings may help explain the increased protection against S. pneumoniae colonization and noninvasive pneumonia and the longer duration of protection against invasive pneumococcal disease, mediated by PCV13.

Notch signaling regulates endothelial progenitor cell activity during recovery from arterial injury in hypercholesterolemic mice.

BACKGROUND: Little is known about the role of endothelial progenitor cells (EPCs) in atherosclerosis. Accordingly, we performed a series of assessments with hypercholesterolemic (apolipoprotein E-null [ApoE(-/-)]) and wild-type (WT) mice to evaluate how cholesterol influences reendothelialization, atherosclerosis, and EPC function after arterial injury. METHODS AND RESULTS: Unexpectedly, reendothelialization (assessed by resistance to Evans blue staining) and circulating EPC counts (EPC culture assay) were greater in ApoE(-/-) mice than in WT mice, and transplantation of ApoE(-/-) bone marrow in WT mice accelerated endothelial recovery and increased recruitment of bone marrow-derived EPCs to the neoendothelium. Cholesterol concentration-dependently promoted the proliferation (MTS assay) of both ApoE(-/-) and WT EPCs, and the concentration dependence of EPC adhesion (to vitronectin-, collagen type I-, fibronectin-, and laminin-coated plates), migration (modified Boyden chamber assay), and antiapoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling stain) activity was biphasic. Cholesterol enhanced the messenger RNA expression (quantitative, real-time reverse-transcription polymerase chain reaction) of vascular endothelial growth factor and inhibited Notch1 messenger RNA expression in both ApoE(-/-) and WT EPCs, whereas endothelial nitric oxide synthase messenger RNA expression increased in ApoE(-/-) EPCs and declined in WT EPCs after cholesterol exposure. EPC activity was greater in Notch1(+/-) EPCs than in WT EPCs, and transplantation of Notch1(+/-) bone marrow accelerated endothelial recovery after arterial injury in WT mice. CONCLUSIONS: The results presented here provide novel insights into the role of EPCs during atherosclerosis and suggest that cholesterol and Notch1 may be involved in the regulation of EPC activity.

Sonic hedgehog induces angiogenesis via Rho kinase-dependent signaling in endothelial cells.

The morphogen Sonic hedgehog (Shh) promotes neovascularization in adults by inducing pro-angiogenic cytokine expression in fibroblasts; however, the direct effects of Shh on endothelial cell (EC) function during angiogenesis are unknown. Our findings indicate that Shh promotes capillary morphogenesis (tube length on Matrigel increased to 271+/-50% of the length in untreated cells, p=0.00003), induces EC migration (modified Boyden chamber assay, 191+/-35% of migration in untreated cells, p=0.00009), and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN) mRNA (real-time RT-PCR), which are essential for Shh-induced angiogenesis both in vitro and in vivo. Shh activity in ECs is mediated by Rho, rather than through the "classic" Shh signaling pathway, which involves the Gli transcription factors. The Rho dependence of Shh-induced EC angiogenic activity was documented both in vitro, with dominant-negative RhoA and Rho kinase (ROCK) constructs, and in vivo, with the ROCK inhibitor Y27632 in the mouse corneal angiogenesis model. Finally, experiments performed in MMP-9- and OPN-knockout mice confirmed the roles of the ROCK downstream targets MMP-9 and OPN in Shh-induced angiogenesis. Collectively, our results identify a "nonclassical" pathway by which Shh directly modulates EC phenotype and angiogenic activity.

T-cell vaccination reduces simian immunodeficiency virus levels in semen.

Recent findings suggest that most sexual transmission of human immunodeficiency virus type 1 (HIV-1) occurs during the acute phase of infection when viral replication is most intense. Here, we show that vaccine-elicited cellular immune responses can significantly reduce simian immunodeficiency virus levels in the semen during the period of primary infection in monkeys. A vaccine that decreases the quantity of HIV-1 in the semen of males during primary infection might decrease HIV-1 transmission in human populations and therefore affect the spread of AIDS.

SARS-CoV-2-specific ELISA development.

Critical to managing the spread of COVID-19 is the ability to diagnose infection and define the acquired immune response across the population. While genomic tests for the novel Several Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) detect the presence of viral RNA for a limited time frame, when the virus is shed in the upper respiratory tract, tests able to define exposure and infection beyond this short window of detectable viral replication are urgently needed. Following infection, antibodies are generated within days, providing a durable read-out and archive of exposure and infection. Several antibody tests have emerged to diagnose SARS-CoV-2. Here we report on a qualified quantitative ELISA assay that displays all the necessary characteristics for high-throughput sample analysis. Collectively, this test offers a quantitative opportunity to define both exposure and levels of immunity to SARS-CoV-2.

Mapping functional humoral correlates of protection against malaria challenge following RTS,S/AS01 vaccination.

Vaccine development has the potential to be accelerated by coupling tools such as systems immunology analyses and controlled human infection models to define the protective efficacy of prospective immunogens without expensive and slow phase 2b/3 vaccine studies. Among human challenge models, controlled human malaria infection trials have long been used to evaluate candidate vaccines, and RTS,S/AS01 is the most advanced malaria vaccine candidate, reproducibly demonstrating 40 to 80% protection in human challenge studies in malaria-naïve individuals. Although antibodies are critical for protection after RTS,S/AS01 vaccination, antibody concentrations are inconsistently associated with protection across studies, and the precise mechanism(s) by which vaccine-induced antibodies provide protection remains enigmatic. Using a comprehensive systems serological profiling platform, the humoral correlates of protection against malaria were identified and validated across multiple challenge studies. Rather than antibody concentration, qualitative functional humoral features robustly predicted protection from infection across vaccine regimens. Despite the functional diversity of vaccine-induced immune responses across additional RTS,S/AS01 vaccine studies, the same antibody features, antibody-mediated phagocytosis and engagement of Fc gamma receptor 3A (FCGR3A), were able to predict protection across two additional human challenge studies. Functional validation using monoclonal antibodies confirmed the protective role of Fc-mediated antibody functions in restricting parasite infection both in vitro and in vivo, suggesting that these correlates may mechanistically contribute to parasite restriction and can be used to guide the rational design of an improved vaccine against malaria.

IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure.

Exposure to Mycobacterium tuberculosis (Mtb) results in heterogeneous clinical outcomes including primary progressive tuberculosis and latent Mtb infection (LTBI). Mtb infection is identified using the tuberculin skin test and interferon-γ (IFN-γ) release assay IGRA, and a positive result may prompt chemoprophylaxis to prevent progression to tuberculosis. In the present study, we report on a cohort of Ugandan individuals who were household contacts of patients with TB. These individuals were highly exposed to Mtb but tested negative disease by IFN-γ release assay and tuberculin skin test, 'resisting' development of classic LTBI. We show that 'resisters' possess IgM, class-switched IgG antibody responses and non-IFN-γ T cell responses to the Mtb-specific proteins ESAT6 and CFP10, immunologic evidence of exposure to Mtb. Compared to subjects with classic LTBI, 'resisters' display enhanced antibody avidity and distinct Mtb-specific IgG Fc profiles. These data reveal a distinctive adaptive immune profile among Mtb-exposed subjects, supporting an expanded definition of the host response to Mtb exposure, with implications for public health and the design of clinical trials.

Publisher Correction: IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure.

In the version of this article originally published, there was an error in the abstract. The word disease should not have been included in the sentence "These individuals were highly exposed to Mtb but tested negative disease by IFN-γ release assay and tuberculin skin test, 'resisting' development of classic LTBI". The sentence should have been "These individuals were highly exposed to Mtb but tested negative by IFN-γ release assay and tuberculin skin test, 'resisting' development of classic LTBI." The error has been corrected in the HTML and PDF versions of this article.

Tumor necrosis factor-alpha receptor p75 is required in ischemia-induced neovascularization.

BACKGROUND: Aging is a risk factor for coronary and peripheral artery disease. Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is expressed in ischemic tissue and is known to modulate angiogenesis. Little is known about the role of TNF-alpha receptors (TNFR1/p55 and TNFR2/p75) in angiogenic signaling. METHODS AND RESULTS: We studied neovascularization in the hindlimb ischemia model in young and old TNFR2/p75 knockout (p75KO) and wild-type age-matched controls. Between days 7 to 10 after hindlimb surgery, 100% of old p75KOs experienced autoamputation of the operated limbs, whereas none of the age-matched wild-type mice exhibited hindlimb necrosis. Poor blood flow recovery in p75KO mice was associated with increased endothelial cell apoptosis, decreased capillary density, and significant reductions in the expression of vascular endothelial growth factor and basic fibroblast growth factor-2 mRNA transcripts in ischemic tissue and in circulating endothelial progenitor cells. The number of circulating bone marrow-derived endothelial progenitor cells was significantly reduced in p75KO mice. Transplantation of wild-type bone marrow mononuclear cells into irradiated old p75KO mice 1 month before hindlimb surgery prevented limb loss. CONCLUSIONS: Our present study suggests that ischemia-induced endothelial progenitor cell-mediated neovascularization is dependent, at least in part, on p75 TNF receptor expressed in bone marrow-derived cells. Specifically, endothelial cell/endothelial progenitor cell survival, vascular endothelial growth factor expression, endothelial progenitor cell mobilization from bone marrow, endothelial progenitor cell differentiation, and ultimately ischemia-induced collateral vessel development are dependent on signaling through TNFR2/p75. Furthermore, because TNFR2/p75 becomes an age-related limiting factor in postischemic recovery, it may be a potential gene target for therapeutic interventions in adult vascular diseases.

The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-alpha-induced transcriptional repression of cyclin A.

TNF-alpha modulates EC proliferation and thereby plays a central role in new blood vessel formation in physiologic and pathologic circumstances. TNF-alpha is known to downregulate cyclin A, a key cell cycle regulatory protein, but little else is known about how TNF-alpha modulates EC cell cycle and angiogenesis. Using primary ECs, we show that ezrin, previously considered to act primarily as a cytoskeletal protein and in cytoplasmic signaling, is a TNF-alpha-induced transcriptional repressor. TNF-alpha exposure leads to Rho kinase-mediated phosphorylation of ezrin, which translocates to the nucleus and binds to cell cycle homology region repressor elements within the cyclin A promoter. Overexpression of dominant-negative ezrin blocks TNF-alpha-induced modulation of ezrin function and rescues cyclin A expression and EC proliferation. In vivo, blockade of ezrin leads to enhanced transplanted EC proliferation and angiogenesis in a mouse hind limb ischemia model. These observations suggest that TNF-alpha regulates angiogenesis via Rho kinase induction of a transcriptional repressor function of the cytoskeletal protein ezrin and that ezrin may represent a suitable therapeutic target for processes dependent on EC proliferation.

Endothelial progenitor thrombospondin-1 mediates diabetes-induced delay in reendothelialization following arterial injury.

Delayed reendothelialization contributes to restenosis after angioplasty and stenting in diabetes. Prior data have shown that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to endothelial recovery after arterial injury. We investigated the hypothesis that the EPC contribution to reendothelialization may be impaired in diabetes, resulting in delayed reendothelialization. Reendothelialization was significantly reduced in diabetic mice compared with nondiabetic mice in a wire-induced carotid denudation model. The EPC contribution to neoendothelium was significantly reduced in Tie2/LacZ BM-transplanted diabetic versus nondiabetic mice. BM from diabetic and nondiabetic mice was transplanted into nondiabetic mice, revealing that reendothelialization was impaired in the recipients of diabetic BM. To examine the relative roles of denuded artery versus EPCs in diabetes, we injected diabetic and nondiabetic EPCs intravenously after arterial injury in diabetic and nondiabetic mice. Diabetic EPCs recruitment to the neoendothelium was significantly reduced, regardless of the diabetic status of the recipient mice. In vitro, diabetic EPCs exhibited decreased migration and adhesion activities. Vascular endothelial growth factor and endothelial NO synthase expressions were also significantly reduced in diabetic EPCs. Notably, thrombospondin-1 mRNA expression was significantly upregulated in diabetic EPCs, associating with the decreased EPC adhesion activity in vitro and in vivo. Reendothelialization is impaired by malfunctioning EPCs in diabetes. Diabetic EPCs have phenotypic differences involving thrombospondin-1 expression compared with nondiabetic EPCs, revealing potential novel mechanistic insights and therapeutic targets to improve reendothelialization and reduce restenosis in diabetes.

Estradiol enhances recovery after myocardial infarction by augmenting incorporation of bone marrow-derived endothelial progenitor cells into sites of ischemia-induced neovascularization via endothelial nitric oxide synthase-mediated activation of matrix metalloproteinase-9.

BACKGROUND: Recent data have indicated that estradiol can modulate the kinetics of endothelial progenitor cells (EPCs) via endothelial nitric oxide synthase (eNOS)-dependent mechanisms. We hypothesized that estradiol could augment the incorporation of bone marrow (BM)-derived EPCs into sites of ischemia-induced neovascularization, resulting in protection from ischemic injury. METHODS AND RESULTS: Myocardial infarction (MI) was induced by ligation of the left coronary artery in ovariectomized mice receiving either 17beta-estradiol or placebo. Estradiol induced significant increases in circulating EPCs 2 and 3 weeks after MI in estradiol-treated animals, and capillary density was significantly greater in estradiol-treated animals. Greater numbers of BM-derived EPCs were observed at ischemic sites in estradiol-treated animals than in placebo-treated animals 1 and 4 weeks after MI. In eNOS-null mice, the effect of estradiol on mobilization of EPCs was lost, as was the functional improvement in recovery from acute myocardial ischemia. A decrease was found in matrix metalloproteinase-9 (MMP-9) expression in eNOS-null mice under basal and estradiol-stimulated conditions after MI, the mobilization of EPCs by estradiol was lost in MMP-9-null mice, and the functional benefit conferred by estradiol treatment after MI in wild-type mice was significantly attenuated. CONCLUSIONS: Estradiol preserves the integrity of ischemic tissue by augmenting the mobilization and incorporation of BM-derived EPCs into sites of neovascularization by eNOS-mediated augmentation of MMP-9 expression in the BM. Moreover, these data have broader implications with regard to our understanding of the role of EPCs in post-MI recovery and on the sex discrepancy in cardiac events.

Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial progenitor cell-mediated microvascular remodeling.

BACKGROUND: Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial-mesenchymal interaction during embryonic development. Recent observations indicate that exogenous administration of Shh can induce angiogenesis and may accelerate repair of ischemic myocardium and skeletal muscle. Because angiogenesis plays a pivotal role in wound repair, we hypothesized that activation of the hedgehog pathway may promote a favorable effect on microvascular remodeling during cutaneous wound healing and thereby accelerate wound closure. Because diabetes is associated with impaired wound healing, we tested this hypothesis in a diabetic model of cutaneous wound repair. METHODS AND RESULTS: In Ptc1-LacZ mice, cutaneous injury resulted in LacZ expression, indicating that expression of the Shh receptor Patched was induced and therefore that the Shh signaling pathway was intact postnatally and upregulated in the process of wound repair. In diabetic mice, topical gene therapy with the use of naked DNA encoding for Shh resulted in significant local gene expression and acceleration of wound recovery. The acceleration in wound healing was notable for increased wound vascularity. In bone marrow transplantation models, the enhanced vascularity of the wound was shown to be mediated, at least in part, by enhanced recruitment of bone marrow-derived endothelial progenitor cells. In vitro, Shh promoted production of angiogenic cytokines from fibroblasts as well as proliferation of dermal fibroblasts. Furthermore, Shh directly promoted endothelial progenitor cell proliferation, migration, adhesion, and tube formation. CONCLUSIONS: These findings suggest that a simple strategy of topically applied Shh gene therapy may have significant therapeutic potential for enhanced wound healing in patients with impaired microcirculation such as occurs in diabetes.

IL-10-induced TNF-alpha mRNA destabilization is mediated via IL-10 suppression of p38 MAP kinase activation and inhibition of HuR expression.

Inflammation plays an essential role in vascular injury and repair. Mononuclear phagocytes are important contributors in these processes, in part, via adhesive interactions and secretion of proinflammatory cytokines. The antiinflammatory cytokine interleukin (IL)-10 suppresses such responses via deactivation of monocytes/macrophages and repression of inflammatory cytokine expression. The mechanisms of IL-10's suppressive action are, however, incompletely characterized. Here, we report that systemic IL-10 treatment after carotid artery denudation in mice blunts inflammatory cell infiltration and arterial tumor necrosis factor (TNF) expression. At the molecular level, in a human monocytic cell line, U937 IL-10 suppressed LPS-induced mRNA expression of a number of inflammatory cytokines, mainly via posttranscriptional mRNA destabilization. Detailed studies on IL-10 regulation of TNF-alpha mRNA expression identified AU-rich elements (ARE) in the 3' untranslated region as a necessary determinant of IL-10-mediated TNF-alpha mRNA destabilization. IL-10 sensitivity to TNF depends on the ability of IL-10 to inhibit the expression and mRNA-stabilizing protein HuR and via IL-10 mediated repression of p38 mitogen-activated protein (MAP) kinase activation. Because IL-10 function and signaling are important components for control of inflammatory responses, these results may provide insights necessary to develop strategies for modulating vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.

Functionally novel tumor necrosis factor-alpha-modulated CHR-binding protein mediates cyclin A transcriptional repression in vascular endothelial cells.

Local expression of tumor necrosis factor-alpha (TNF-alpha) at the sites of arterial injury after balloon angioplasty, suppresses endothelial cell (EC) proliferation and negatively affects reendothelialization of the injured vessel. We have previously reported that in vitro exposure of ECs to TNF-alpha induced EC growth arrest and apoptosis. These effects were mediated, at least in part, by downregulation of cell cycle regulatory proteins. In the present study, we report potential mechanism(s) for TNF-alpha-mediated suppression of cyclin A in ECs. TNF-alpha exposure to ECs completely abrogated cyclin A mRNA expression via mechanisms involving both transcriptional and posttranscriptional modifications. TNF-alpha inhibited de novo cyclin A mRNA synthesis and suppressed cyclin A promoter activity. Utilizing deletion mutants of human cyclin A promoter, we have identified CDE-CHR (Cell cycle-Dependent Elements-Cell cycle genes Homology Region) region of cyclin A promoter as a target for TNF-alpha suppressive action. Experiments to investigate CDE-CHR binding proteins/factors revealed a TNF-alpha-mediated increase in specific DNA binding activity to the CHR elements. This increase in binding activity by TNF-alpha was mediated via the induction of a functionally novel 84-kDa protein that binds specifically to CHR in Southwestern assays. UV cross-linking and SDS-PAGE analysis of proteins eluted from specific complex confirmed the presence of this 84-kDa protein. Moreover, induction of this protein by TNF-alpha was protein synthesis dependent. Additionally, exposure of ECs to TNF-alpha markedly reduced cyclin A mRNA stability. Targeted disruption of this protein could potentially be a therapeutic strategy to rescue EC proliferation in vivo.

Tumor necrosis factor-mediated E2F1 suppression in endothelial cells: differential requirement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signal transduction pathways.

After balloon angioplasty, locally expressed tumor necrosis factor (TNF)-alpha disrupts endothelial cell (EC) proliferation and reendothelialization of the injured vessel. We have previously reported that TNF inhibits the EC cycle and downregulates the transcription factor E2F1. Ectopic expression of E2F1 at the site of injury improves reendothelialization of the injured vessel. In this study, we report that c-Jun N-terminal kinase (JNK) 1 and p38 mitogen-activated protein kinases (MAPKs) are differentially required for E2F1 expression and activity in ECs. Overexpression of constitutively active JNK1 mimicked TNF-mediated inhibitory events, whereas dominant-negative JNK1 prevented these effects. E2F cis elements in the promoter of E2F1 gene mediate suppressive actions of TNF, because removal of these sites rendered E2F1 promoter activity insensitive to TNF. JNK1 physically interacted with E2F1 and inactivated it via direct phosphorylation. Additionally, TNF inhibited Rb phosphorylation and dissociation from E2F1. Overexpression of constitutively active p38 MAPK facilitated Rb-E2F1 dissociation, whereas that of dominant-negative p38 MAPK did not. Taken together, these data suggest a differential requirement of JNK1 and p38 MAPK in TNF regulation of E2F1. Targeted inactivation of JNK1 at arterial injury sites may represent a potential therapeutic intervention for ameliorating TNF-mediated EC dysfunction.

Engineering the response to vascular injury: divergent effects of deregulated E2F1 expression on vascular smooth muscle cells and endothelial cells result in endothelial recovery and inhibition of neointimal growth.

Tumor necrosis factor-alpha (TNF-alpha) is expressed locally in the vessel wall after angioplasty and induces growth arrest and apoptosis in endothelial cells (ECs), thereby delaying reendothelialization. Prior studies have shown that direct antagonism of TNF-alpha, using a systemically administered soluble receptor, can enhance endothelial recovery and reduce neointimal thickening. These studies have also shown that downregulation of the transcription factor E2F1 was a key mechanism of TNF's effect on ECs. We now show that Ad-E2F1 overexpression at sites of balloon injury accelerates functional endothelial recovery, consistent with the prior in vitro findings. Moreover these studies also reveal divergent effects of TNF-alpha and overexpression of E2F1 on ECs versus VSMCs. TNF-alpha exposure of VSMCs had no affect on proliferation or apoptosis, in contrast to the effect seen in ECs. In Ad-E2F1-transduced VSMCs, however, TNF-alpha-induced marked apoptosis in contrast to the survival effect seen in ECs. Finally, these studies suggest that differential activation of NF-kappaB may play a key role in mediating these opposing effects. Nuclear translocation and transcriptional activity of NF-kappaB was markedly attenuated in Ad-E2F1-transduced VSMCs, whereas it remained active in similarly treated ECs when the cells were exposed to TNF-alpha. These studies reveal that overexpression of Ad-E2F1 primes VSMCs to TNF-alpha-induced apoptosis. Furthermore, E2F1 potentiates VSMC death by blocking antiapoptotic signaling pathway through inhibition of NF-kappaB activation. The divergent responses of VSMCs and ECs to E2F1 overexpression provide unique therapeutic possibilities: simultaneously targeting the cell cycle of two different cell types, within same tissue microenvironment resulting in opposite and biologically complimentary effects.