Guidelines on the use of sex and gender in cardiovascular research.

In cardiovascular research, sex and gender have not typically been considered in research design and reporting until recently. This has resulted in clinical research findings from which not only all women, but also gender-diverse individuals have been excluded. The resulting dearth of data has led to a lack of sex- and gender-specific clinical guidelines and raises serious questions about evidence-based care. Basic research has also excluded considerations of sex. Including sex and/or gender as research variables not only has the potential to improve the health of society overall now, but it also provides a foundation of knowledge on which to build future advances. The goal of this guidelines article is to provide advice on best practices to include sex and gender considerations in study design, as well as data collection, analysis, and interpretation to optimally establish rigor and reproducibility needed to inform clinical decision-making and improve outcomes. In cardiovascular physiology, incorporating sex and gender is a necessary component when optimally designing and executing research plans. The guidelines serve as the first guidance on how to include sex and gender in cardiovascular research. We provide here a beginning path toward achieving this goal and improve the ability of the research community to interpret results through a sex and gender lens to enable comparison across studies and laboratories, resulting in better health for all.

Distinct hypoxia-induced translational profiles of embryonic and adult-derived macrophages.

Tissue resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Posttranscriptional regulation determining mRNA stability and translation rate may override transcriptional signals in response to hypoxia. We profiled differentially regulated BMDM and FLDM transcripts in response to hypoxia at the level of mRNA translation. Using a translating ribosome affinity purification (TRAP) assay and RNA-seq, we identified non-overlapping transcripts with increased translation rate in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2) after hypoxia. We further identified hypoxia-induced transcripts within these subsets that are regulated by the RNA-binding protein HuR. These findings define translational differences in macrophage subset gene expression programs, highlighting potential therapeutic targets in ischemic myocardium.

Effects of androgen excess and body mass index on endothelial function in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is associated with endothelial dysfunction; whether this is attributable to comorbid hyperandrogenism and/or obesity remains to be established. Therefore, we 1) compared endothelial function between lean and overweight/obese (OW/OB) women with and without androgen excess (AE)-PCOS and 2) examined androgens as potential modulators of endothelial function in these women. The flow-mediated dilation (FMD) test was applied in 14 women with AE-PCOS (lean: n = 7; OW/OB: n = 7) and 14 controls (CTRL; lean: n = 7, OW/OB: n = 7) at baseline (BSL) and following 7 days of ethinyl estradiol supplementation (EE; 30 µg/day) to assess the effect of a vasodilatory therapeutic on endothelial function; at each time point we assessed peak increases in diameter during reactive hyperemia (%FMD), shear rate, and low flow-mediated constriction (%LFMC). BSL %FMD was attenuated in lean AE-PCOS versus both lean CTRL (5.2 ± 1.5 vs. 10.3 ± 2.6%, P < 0.01) and OW/OB AE-PCOS (5.2 ± 1.5 vs. 6.6 ± 0.9%, P = 0.048). A negative correlation between BSL %FMD and free testosterone was observed in lean AE-PCOS only (R2 = 0.68, P = 0.02). EE increased %FMD in both OW/OB groups (CTRL: 7.6 ± 0.6 vs. 10.4 ± 2.5%, AE-PCOS: 6.6 ± 0.9 vs. 9.6 ± 1.7%, P < 0.01), had no impact on %FMD in lean AE-PCOS (5.17 ± 1.5 vs. 5.17 ± 1.1%, P = 0.99), and reduced %FMD in lean CTRL (10.3 ± 2.6 vs. 7.6 ± 1.2%, P = 0.03). Collectively, these data indicate that lean women with AE-PCOS exhibit more severe endothelial dysfunction than their OW/OB counterparts. Furthermore, endothelial dysfunction appears to be mediated by circulating androgens in lean but not in OW/OB AE-PCOS, suggesting a difference in the endothelial pathophysiology of AE-PCOS between these phenotypes.NEW & NOTEWORTHY We present evidence for marked endothelial dysfunction in lean women with androgen excess polycystic ovary syndrome (AE-PCOS) that is 1) associated with free testosterone levels, 2) impaired relative to overweight/obese women with AE-PCOS, and 3) unchanged following short-term ethinyl estradiol supplementation. These data indicate an important direct effect of androgens on the vascular system in women with AE-PCOS. Our data also suggest that the relationship between androgens and vascular health differs between phenotypes of AE-PCOS.

Assessing the Impact of the American Heart Association's Research Portfolio: A Scientific Statement From the American Heart Association.

A task force composed of American Heart Association (AHA) Research Committee members established processes to measure the performance of the AHA's research portfolio and evaluated key outcomes that are fundamental to the overall success of the program. This report reviews progress that the AHA research program has had in achieving its goals relevant to the research programs in the AHA's research portfolio from 2008 to 2017. Comprehensive performance metrics were identified to assess the impact of AHA funding on researchers' career progress and research outcomes. Metrics included bibliometric analysis (ie, tracking of publications and their impact) and career development measures (ie, subsequent grant funding, intellectual property, faculty appointment/promotion, or industry position). Publication rates ranged from ≈0.5 to 4 publications per year, with a strong correlation between number of publications per year and later career stage. The Field-Weighted Citation Index, a metric of bibliometric impact, was between 1.5 and 3.0 for all programs, indicating that AHA awardee publications had a higher citation impact compared with similar publications. To gain insight into the career progression of AHA awardees, a 2-year postaward survey was distributed. Of the Postdoctoral Fellowship recipient respondents, 72% obtained academic research positions, with the remaining working in industry or government research settings; 72% of those in academic positions obtained additional funding. Among respondents who were Beginning Grant-in-Aid and Scientist Development Grant awardees, 45% received academic promotions and 83% obtained additional funding. Measuring performance of the AHA's research portfolio is critical to ensure that its strategic goals are met and to show the AHA's commitment to high-quality, impactful research.

Androgens drive microvascular endothelial dysfunction in women with polycystic ovary syndrome: role of the endothelin B receptor.

KEY POINTS: Polycystic ovary syndrome (PCOS) is a complex syndrome with cardiovascular risk factors, including obesity and insulin resistance. PCOS is also associated with high androgens, increases the risk of cardiovascular dysfunction in women. Due to the complexity of PCOS, had it has been challenging to isolate specific causes of the cardiovascular dysfunction. Our measure of cardiovascular dysfunction (endothelial dysfunction) was most profound in lean women with PCOS. The endothelin-1-induced vasodilation in these PCOS subject, was dependent on the ETB R but was not NO-dependent. We also demonstrated oestrogen administration improved endothelial function in lean and obese women with PCOS likely because oestrogen increased NO availability. Our studies indicate a primary role for androgens in cardiovascular dysfunction in PCOS. ABSTRACT: Endothelin-1 (ET-1) is an indicator of endothelial injury and dysfunction and is elevated in women with androgen excess polycystic ovary syndrome (AE-PCOS). The endothelin B receptor (ETB R) subtype mediates vasodilatation, but is blunted in women with PCOS. We hypothesized that androgen drives endothelial dysfunction in AE-PCOS women and oestradiol (EE) administration reverses these effects. We assessed microvascular endothelial function in women with (7 lean and 7 obese) and without AE-PCOS (controls, 6 lean, 7 obese). Only obese AE-PCOS women were insulin resistant (IR). We evaluated cutaneous vascular conductance (%CVCmax ) with laser Doppler flowmetry during low dose intradermal microdialysis ET-1 perfusions (1, 3, 4, 5 and 7 pmol) with either lactated Ringer solution alone, or with ETB R (BQ-788), or nitric oxide (NO) inhibition (l-NAME). Log[ET-1]-%maxCVC dose-response curves demonstrated reduced vasodilatory responses to ET-1 in lean AE-PCOS (logED50 , 0.59 ± 0.08) versus lean controls (logED50 , 0.49 ± 0.09, P < 0.05), but not compared to obese AE-PCOS (logED50 , 0.65 ± 0.09). ETB R inhibition decreased ET-1-induced vasodilatation in AE-PCOS women (logED50 , 0.64 ± 0. 22, P < 0.05). This was mechanistically observed at the cellular level, with ET-1-induced, DAF-FM-measurable endothelial cell NO production, which was abrogated by dihydrotestosterone in an androgen receptor-dependent manner. EE augmented the cutaneous vasodilating response to ET-1(logED50 0.29 ± 0.21, 0.47 ± 0.09, P < 0.05 for lean and obese, respectively). Androgens drive endothelial dysfunction in lean and obese AE-PCOS. We propose that the attenuated ET-1-induced vasodilatation in AE-PCOS is a consequence of androgen receptor-mediated, suppressed ETB R-stimulated NO production, and is reversed with EE.

T cell LFA-1-induced proinflammatory mRNA stabilization is mediated by the p38 pathway kinase MK2 in a process regulated by hnRNPs C, H1 and K.

Activation of the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator.

Rac2 Modulates Atherosclerotic Calcification by Regulating Macrophage Interleukin-1ß Production.

OBJECTIVE: The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. APPROACH AND RESULTS: Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1ß (IL-1ß) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1ß expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1ß levels. Moreover, we found that elevated IL-1ß was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. CONCLUSIONS: Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1ß expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.

The molecular actions of oestrogen in the regulation of vascular health.

NEW FINDINGS: What is the topic of this review? This review summarizes the beneficial actions of oestrogen on the vasculature, highlighting both molecular mechanisms and functional outcomes. What advances does it highlight? The net effect of oestrogen on the vascular health of women continues to be debated. Recent advances have provided strong evidence for the role of membrane-bound oestrogen receptors in the maintenance of normal endothelial function. On a broader scale, functional outcomes of oestrogen actions on the vasculature may mediate the reduced risk of cardiovascular disease in premenopausal women. The conflicting implications of the large-scale clinical menopausal hormone therapy trials in humans versus the findings of studies on experimental animals underscore the limitations within our understanding of the molecular actions of oestrogen. However, recent research has provided improved insight into the actions of oestrogen on the endothelium and vascular smooth muscle. This review outlines the actions of oestrogen as it contributes to vascular structure, function and health.

Menopause and the Heart.

Cardiovascular disease is the leading cause of death in postmenopausal US women. The contribution of postmenopausal hormone replacement therapy to cardiovascular risk is one of the most controversial women's health topics. Strikingly discordant results, between observational and randomized clinical trials, have been reported. Remaining questions regarding time of hormone therapy initiation are discussed, as are ongoing trials focused on these questions. Cardiovascular concerns, cautions, and current recommendations for use are delineated.

Chemokine-coupled ß2 integrin-induced macrophage Rac2-Myosin IIA interaction regulates VEGF-A mRNA stability and arteriogenesis.

Myeloid cells are important contributors to arteriogenesis, but their key molecular triggers and cellular effectors are largely unknown. We report, in inflammatory monocytes, that the combination of chemokine receptor (CCR2) and adhesion receptor (ß2 integrin) engagement leads to an interaction between activated Rac2 and Myosin 9 (Myh9), the heavy chain of Myosin IIA, resulting in augmented vascular endothelial growth factor A (VEGF-A) expression and induction of arteriogenesis. In human monocytes, CCL2 stimulation coupled to ICAM-1 adhesion led to rapid nuclear-to-cytosolic translocation of the RNA-binding protein HuR. This activation of HuR and its stabilization of VEGF-A mRNA were Rac2-dependent, and proteomic analysis for Rac2 interactors identified the 226 kD protein Myh9. The level of induced Rac2-Myh9 interaction strongly correlated with the degree of HuR translocation. CCL2-coupled ICAM-1 adhesion-driven HuR translocation and consequent VEGF-A mRNA stabilization were absent in Myh9(-/-) macrophages. Macrophage VEGF-A production, ischemic tissue VEGF-A levels, and flow recovery to hind limb ischemia were impaired in myeloid-specific Myh9(-/-) mice, despite preserved macrophage recruitment to the ischemic muscle. Micro-CT arteriography determined the impairment to be defective induced arteriogenesis, whereas developmental vasculogenesis was unaffected. These results place the macrophage at the center of ischemia-induced arteriogenesis, and they establish a novel role for Myosin IIA in signal transduction events modulating VEGF-A expression in tissue.

The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes.

Inflammatory cells acquire a polarized phenotype to migrate towards sites of infection or injury. A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization. However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo. Using genetically encoded bioprobes and high-resolution live imaging, we reveal the existence of F-actin oscillations in the trailing edge and constant repositioning of the microtubule organizing center (MTOC) to direct leukocyte migration in wounded medaka fish larvae (Oryzias latipes). Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both the generation of traction force and the directionality of leukocyte migration to sites of injury.

Silver lining in the dark cloud of aneurysm disease.

An ascending aortic aneurysm is a common and very much unwelcome diagnosis that has never been associated with anything positive. We believe, however, that there actually is a silver lining to this disease: aortic root and ascending aortic aneurysms actually protect against atherosclerosis. We have found that patients with ascending aneurysms have both decreased arterial calcification and carotid intima-media thickness, late and early indicators of atherosclerosis, respectively. In addition to these clinical data, we also found data regarding molecular mechanisms, genetic studies, and pharmacologic evidence that corroborate our clinical findings, in particular, evidence regarding matrix metalloproteinases and transforming growth factor-ß pathways. In this article, we lay out the evidence that has been accruing for the protective effect of ascending aneurysms against atherosclerosis.

Ceramide-activated phosphatase mediates fatty acid-induced endothelial VEGF resistance and impaired angiogenesis.

Endothelial dysfunction, including endothelial hyporesponsiveness to prototypical angiogenic growth factors and eNOS agonists, underlies vascular pathology in many dysmetabolic states. We investigated effects of a saturated free fatty acid, palmitic acid (PA), on endothelial cell responses to VEGF. PA-pretreated endothelial cells had markedly diminished Akt, eNOS, and ERK activation responses to VEGF, despite normal VEGFR2 phosphorylation. PA inhibited VEGF-induced angiogenic cord formation in Matrigel, and PA-treated endothelial cells accumulated early species (C16) ceramide. The serine palmitoyltransferase inhibitor myriocin reversed these defects. Protein phosphatase 2A (PP2A) became more eNOS-associated in PA-treated cells; the PP2A inhibitor okadaic acid reversed PA-induced signaling defects. Mice fed a diet high in saturated fat for 2 to 3 weeks had impaired i) aortic Akt and eNOS phosphorylation to infused VEGF, ii) ear angiogenic responses to intradermal adenoviral-VEGF injection, and iii) vascular flow recovery to hindlimb ischemia as indicated by laser Doppler and αVß3 SPECT imaging. High-fat feeding did not impair VEGF-induced signaling or angiogenic responses in mice with reduced serine palmitoyltransferase expression. Thus, de novo ceramide synthesis is required for these detrimental PA effects. The findings demonstrate an endothelial VEGF resistance mechanism conferred by PA, which comprises ceramide-induced, PP2A-mediated dephosphorylation of critical activation sites on enzymes central to vascular homeostasis and angiogenesis. This study defines potential molecular targets for preservation of endothelial function in metabolic syndrome.

Endothelial estrogen receptor isoforms and cardiovascular disease.

Rapid, nongenomic vascular cell and tissue responses to estrogen have been demonstrated for more than a decade. Although the pendulum continues to swing, accumulating evidence, both clinical and pre-clinical, support favorable effects of ovarian steroid hormones in the vascular system. These effects are mediated both by classical steroid hormone receptor-mediated transcriptional modulation, and largely by endothelial plasma membrane-associated estrogen receptor (ER)α, which when engaged triggers a signaling cascade resulting in release of cardioprotective nitric oxide (NO). In addition to full-length ERα (ER66), an N-terminus truncated ERα isoform, ER46, plays a key role in these rapid endothelial responses to 17ß-estradiol (E2). We have recently determined that ER46 can be a Type I integral transmembrane molecule. In this review, we discuss ER isoforms, rapid E2-stimulated signaling in the endothelium, the importance of the ER46 transmembrane orientation, and the clinical context of this rapid endothelial signaling.

Transmembrane protein ESDN promotes endothelial VEGF signaling and regulates angiogenesis.

Aberrant blood vessel formation contributes to a wide variety of pathologies, and factors that regulate angiogenesis are attractive therapeutic targets. Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a neuropilin-related transmembrane protein expressed in ECs; however, its potential effect on VEGF responses remains undefined. Here, we generated global and EC-specific Esdn knockout mice and demonstrated that ESDN promotes VEGF-induced human and murine EC proliferation and migration. Deletion of Esdn in the mouse interfered with adult and developmental angiogenesis, and knockdown of the Esdn homolog (dcbld2) in zebrafish impaired normal vascular development. Loss of ESDN in ECs blunted VEGF responses in vivo and attenuated VEGF-induced VEGFR-2 signaling without altering VEGF receptor or neuropilin expression. Finally, we found that ESDN associates with VEGFR-2 and regulates its complex formation with negative regulators of VEGF signaling, protein tyrosine phosphatases PTP1B and TC-PTP, and VE-cadherin. These findings establish ESDN as a regulator of VEGF responses in ECs that acts through a mechanism distinct from neuropilins. As such, ESDN may serve as a therapeutic target for angiogenesis regulation.

Molecular imaging of vascular endothelial growth factor receptors in graft arteriosclerosis.

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis. Graft arteriosclerosis is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors and vascular remodeling in human coronary artery grafts by molecular imaging. METHODS AND RESULTS: VEGF receptor specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells led to significant neointima formation in the grafts over a period of 4 weeks. Near-infrared fluorescent imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homolog of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGF receptor-1 but not VEGF receptor-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area. CONCLUSIONS: Molecular imaging of VEGF receptors may provide a noninvasive tool for detection of graft arteriosclerosis in solid organ transplantation.

Macrophage ß2 integrin-mediated, HuR-dependent stabilization of angiogenic factor-encoding mRNAs in inflammatory angiogenesis.

HuR is a member of the Drosophila Elav protein family that binds mRNA degradation sequences and prevents RNase-mediated degradation. Such HuR-mediated mRNA stabilization, which is stimulated by integrin engagement and is controlled at the level of HuR nuclear export, is critically involved in T-cell cytokine production. However, HuR's role in macrophage soluble factor production, in particular in response to angiogenic stimuli, has not yet been established. We show that the labile transcripts that encode vascular endothelial growth factor and matrix metalloproteinase-9 are stabilized when murine macrophages adhere to the ß(2) integrin ligand intercellular adhesion molecule-1. This mRNA stabilization response was absent in bone marrow-derived macrophages obtained from conditional macrophage-specific HuR knockout mice. The microvascular angiogenic response to an inflammatory stimulus (ie, subcutaneous polyvinyl alcohol sponge implantation) was markedly diminished in these macrophage HuR knockout mice despite the equal levels of macrophage localization to those observed in littermate wild-type controls. Furthermore, blood flow recovery and ischemic muscle neovascularization after femoral artery ligation were impaired in the conditional macrophage-specific HuR knockout mice. These results demonstrate that dynamic effects on mRNA, mediated by the RNA-binding and RNA-stabilizing protein HuR, are required for macrophage production of angiogenic factors, which play critical roles in the neovascular responses to a variety of stimuli, including tissue ischemia.

Splice isoform estrogen receptors as integral transmembrane proteins.

In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus-truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element-luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets.

VEGF blockade inhibits lymphocyte recruitment and ameliorates immune-mediated vascular remodeling.

RATIONALE: There are conflicting data on the effects of vascular endothelial growth factor (VEGF) in vascular remodeling. Furthermore, there are species-specific differences in leukocyte and vascular cell biology and little is known about the role of VEGF in remodeling of human arteries. OBJECTIVE: We sought to address the role of VEGF blockade on remodeling of human arteries in vivo. METHODS AND RESULTS: We used an anti-VEGF antibody, bevacizumab, to study the effect of VEGF blockade on remodeling of human coronary artery transplants in severe combined immunodeficient mice. Bevacizumab ameliorated peripheral blood mononuclear cell-induced but not interferon-gamma-induced neointimal formation. This inhibitory effect was associated with a reduction in graft T-cell accumulation without affecting T-cell activation. VEGF enhanced T-cell capture by activated endothelium under flow conditions. The VEGF effect could be recapitulated when a combination of recombinant intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 rather than endothelial cells was used to capture T cells. A subpopulation of CD3+ T cells expressed VEGF receptor (VEGFR)-1 by immunostaining and FACS analysis. VEGFR-1 mRNA was also detectable in purified CD4+ T cells and Jurkat and HSB-2 T-cell lines. Stimulation of HSB-2 and T cells with VEGF triggered downstream ERK phosphorylation, demonstrating the functionality of VEGFR-1 in human T cells. CONCLUSIONS: VEGF contributes to vascular remodeling in human arteries through a direct effect on human T cells that enhances their recruitment to the vessel. These findings raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling.

T cell LFA-1 engagement induces HuR-dependent cytokine mRNA stabilization through a Vav-1, Rac1/2, p38MAPK and MKK3 signaling cascade.

BACKGROUND: Engagement of the ß2 integrin, lymphocyte function-associated antigen-1 (LFA-1), results in stabilization of T cell mRNA transcripts containing AU-rich elements (AREs) by inducing rapid nuclear-to-cytosolic translocation of the RNA-stabilizing protein, HuR. However, little is known regarding integrin-induced signaling cascades that affect mRNA catabolism. This study examines the role of the GTPases, Rac 1 and Rac 2, and their downstream effectors, in the LFA-1-induced effects on mRNA. METHODOLOGY/PRINCIPAL FINDINGS: Engagement of LFA-1 to its ligand, ICAM-1, in human peripheral T cells resulted in rapid activation of Rac1 and Rac2. siRNA-mediated knockdown of either Rac1 or Rac2 prevented LFA-1-stimulated stabilization of the labile transcripts encoding IFN-γ and TNF-α, and integrin mediated IFN-γ mRNA stabilization was absent in T cells obtained from Rac2 gene-deleted mice. LFA-1 engagement-induced translocation of HuR and stabilization of TNF- α mRNA was lost in Jurkat cells deficient in the Rac guanine nucleotide exchange factor Vav-1 (J.Vav1). The transfection of J.Vav1 cells with constitutively active Rac1 or Rac2 stabilized a labile ß-globin reporter mRNA, in a HuR-dependent manner. Furthermore, LFA-1-mediated mRNA stabilization and HuR translocation in mouse splenic T cells was dependent on the phosphorylation of the mitogen-activated protein kinase kinase, MKK3, and its target MAP kinase p38MAPK, and lost in T cells obtained from MKK3 gene-deleted mice. CONCLUSIONS/SIGNIFICANCE: Collectively, these results demonstrate that LFA-1-induced stabilization of ARE-containing mRNAs in T cells is dependent on HuR, and occurs through the Vav-1, Rac1/2, MKK3 and p38MAPK signaling cascade. This pathway constitutes a molecular switch that enhances immune and pro-inflammatory gene expression in T cells undergoing adhesion at sites of activation and effector function.