All animal procedures were approved by the Lovelace Respiratory Research Institutes Animal Care and Use Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. were further confirmed using the HIV transgenic (Tg) rats. These animals not only demonstrated increased expression of ICAM-1 mRNA, with a concomitant reduction in the expression of miR-221 in the aorta and heart, but also had increased expression of the ICAM-1 protein that was predominantly in the endothelial cell layer. Taken together, these findings implicate that Tat-mediated induction of ICAM-1 expression plays a critical role in monocyte adhesion observed in HIV-1-associated cardiomyopathies. Introduction Several postmortem studies on AIDS patients have shown clear signs of cardiomyopathies [1]. Clinical studies on HIV-infected patients also provide evidence of progressive cardiac complications following HIV-1 infection [2], [3]. While the advent of anti-retroviral therapy has decreased the incidence of HIV-1 cardiomyopathy (HIVCM), its prevalence is actually on a rise. The mechanism(s) by which HIV-1 induces inflammation of the heart are not well understood, but are likely multifactorial in nature. HIV-1 viral protein Tat that is released by infected monocytes and taken up by neighboring cells has been shown to facilitate interaction of the monocyte with the endothelium [4], [5], resulting in the recruitment of Rabbit Polyclonal to OR4F4 monocytes into the extravascular tissue. This process, in turn, contributes to destruction of the tissue parenchyma and cellular architecture, a classic feature observed in patients with AIDS [5]. Accumulation of monocytes within the tissue leads to tissue damage and dysfunction. Monocyte adhesion is a dynamic, multistep process involving initial rolling of cells along the vessel endothelium in response to inflammatory mediators, arrest to endothelium and subsequent strong adhesion to the systemic vasculature [6]. Interaction of endothelial adhesion molecules with their cognate ligands on monocytes is critical for this process. Up-regulation of adhesion molecules such as ICAM-1 and VCAM-1 is pivotal in the development of inflammatory responses. A previous study has demonstrated that interactions between ICAM-1 expressed on endothelial cells and circulating monocytes may be critical for the adhesion of these cells on the vascular endothelium [7]. HIV Tat is known to exhibit diverse functional aberrations on the endothelial cells [5]. For example, Tat-mediated impaired expression of adhesion molecules has been implicated as an early step in the development of cardiovascular disease associated with HIV-1-infection [5], [8], [9]. Although it has been documented that Tat mediated increase expression of ICAM-1 and VCAM-1 in HUVECs [4], detailed mechanisms underlying this process are not well elucidated. MicroRNAs (miRNAs) are small RNA regulators (18C23 nucleotides) that play essential roles in a wide spectrum of biological processes [10], [11]. These molecules target mRNAs on the basis of complementary sequences between the miRNAs and the 3-untranslated regions (3UTRs) of the target mRNAs, resulting in suppression of cellular target genes by inducing either mRNA degradation and/or translational suppression [11]. Because miRNAs appear to provide quantitative regulation of genes, rather than on-off decisions, they can be envisioned as fine tuners of the cellular responses to external influences [12]. miRNAs regulate many disparate processes including regulation of expression of cell adhesion molecules. Previous reports indicate the role of miR-221 in suppressing ICAM-1 translation and regulating IFN–induced ICAM-1 expression in human cholangiocytes [13]. However, the role of these miRNAs in the context of HIV-1 infection in HUVECs has not been yet determined. The present study was aimed at exploring the molecular mechanisms by which Tat mediates induction of ICAM-1 in vascular endothelial cells. Understanding the regulation of ICAM-1 expression by Tat may provide insights into the development of therapeutic targets aimed at blocking inflammation in the heart of HIV-1.ICAM-1 expression is indicated by red fluorescence, blue fluorescence is nuclei staining (DAPI), and arrows indicate increased ICAM-1 fluorescence. ICAM-1 was regulated by microRNA (miRNA) miR-221 and to some extent by miR-222, both of which are known to target ICAM-1. Functional inhibition of the respective miRNAs with anti-miR oligonucleotides resulted in induction of ICAM-1 protein in HUVECs. Furthermore, Tat-stimulated regulation of ICAM-1 via miR-221/-222 involved the NF-kB-dependent pathway. Functional implication and specificity of up-regulated ICAM-1 was confirmed using the ICAM-1 neutralizing antibody in the cell adhesion assays. These findings were further confirmed using the HIV transgenic (Tg) rats. These animals not only demonstrated increased expression of ICAM-1 mRNA, with a HSF1A concomitant reduction in the expression of miR-221 in the aorta and heart, but also had increased expression of the ICAM-1 protein that was predominantly in the endothelial cell layer. Taken together, these findings implicate that Tat-mediated induction of ICAM-1 expression plays a critical role in monocyte adhesion observed in HIV-1-associated cardiomyopathies. Introduction Several postmortem studies on AIDS patients have shown clear signs of cardiomyopathies [1]. Clinical studies on HIV-infected patients also provide evidence of progressive cardiac complications following HIV-1 infection [2], [3]. While the advent of anti-retroviral therapy has decreased the incidence of HIV-1 cardiomyopathy (HIVCM), its prevalence is actually on a rise. The mechanism(s) by which HIV-1 induces inflammation of the heart are not well understood, but are likely multifactorial in nature. HIV-1 viral protein Tat that is released by infected monocytes and taken up by neighboring cells has been shown to facilitate interaction of the monocyte with the endothelium [4], [5], resulting in the recruitment of monocytes into the extravascular tissue. This process, in turn, contributes to destruction of the tissue parenchyma and cellular architecture, a classic feature observed in patients with AIDS [5]. Accumulation of monocytes within the tissue leads to tissue damage and dysfunction. Monocyte adhesion is a dynamic, multistep process involving initial rolling of cells along the vessel endothelium in response to inflammatory mediators, arrest to endothelium and subsequent strong adhesion to the systemic vasculature [6]. Interaction of endothelial adhesion molecules with their cognate ligands on monocytes is critical for this process. Up-regulation of adhesion HSF1A molecules such as ICAM-1 and VCAM-1 is pivotal in the development of inflammatory responses. A previous study has demonstrated that interactions between ICAM-1 expressed on endothelial cells and circulating monocytes may be critical for the adhesion of these cells on the vascular endothelium [7]. HIV Tat is known to exhibit diverse functional aberrations on the endothelial cells [5]. For example, Tat-mediated impaired expression of adhesion molecules has been implicated as an early step in the development of cardiovascular disease associated with HIV-1-infection [5], [8], [9]. Although it has been documented that Tat mediated increase expression of ICAM-1 and VCAM-1 in HUVECs [4], detailed mechanisms underlying this process are not well elucidated. MicroRNAs (miRNAs) are small RNA regulators (18C23 nucleotides) that play essential roles in a wide spectrum of biological processes [10], [11]. These molecules target mRNAs on the basis of complementary sequences between the miRNAs and the 3-untranslated regions (3UTRs) of the target mRNAs, resulting in suppression of cellular target genes by inducing either mRNA degradation and/or translational suppression [11]. Because miRNAs appear to provide HSF1A quantitative regulation of genes, rather than on-off decisions, they can be envisioned as fine tuners of the cellular responses to external influences [12]. miRNAs regulate many disparate processes including regulation of expression of cell adhesion molecules. Previous reports indicate the role of miR-221 in suppressing ICAM-1 translation and regulating IFN–induced ICAM-1 expression in human cholangiocytes [13]. However, the role of these miRNAs in the context of HIV-1 infection in HUVECs has not been yet determined. The present study was aimed at exploring the molecular mechanisms by which Tat mediates induction of ICAM-1 in vascular endothelial cells. Understanding the regulation of ICAM-1 HSF1A expression by Tat may provide insights into the development of.