HIV and Bone Disease: A Perspective of the Role of microRNAs in Bone Biology upon HIV Infection

Increased life expectancy and the need for long-term antiretroviral therapy have brought new challenges to the clinical management of HIV-infected individuals. The prevalence of osteoporosis and fractures is increased in HIV-infected patients; thus optimal strategies for risk management and treatment in this group of patients need to be defined. Prevention of bone loss is an important component of HIV care as the HIV population grows older. Understanding the mechanisms by which HIV infection affects bone biology leading to osteoporosis is crucial to delineate potential adjuvant treatments. This review focuses on HIV-induced osteoporosis within the context of microRNAs (miRNAs) by reviewing first basic concepts of bone biology as well as current knowledge of the role of miRNAs in bone development. Evidence that HIV-associated osteoporosis is in part independent of therapies employed to treat HIV (HAART) is supported by cross-sectional and longitudinal studies and is the focus of this review.</jats:p

Altered Platelet-Megakaryocyte Endocytosis and Trafficking of Albumin and Fibrinogen in Runx1 Haplodeficiency

Platelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1-haplodeficiency

Transcription Factor RUNX1 Regulates Coagulation Factor XIII-A (F13A1): Decreased Platelet-Megakaryocyte F13A1 Expression and Clot Contraction in RUNX1 Haplodeficiency

BACKGROUND: Germline RUNX1 haplodeficiency (RHD) is associated with thrombocy- topenia, platelet dysfunction, and predisposition to myeloid malignancies. Platelet expression profiling of an RHD patient showed decreased F13A1, encoding for the A subunit of factor (F)XIII, a transglutaminase that cross-links fibrin and induces clot stabilization. FXIII-A is synthesized by hematopoietic cells, megakaryocytes, and monocytes. OBJECTIVES: o understand RUNX1 regulation of F13A1 expression in platelets/mega- karyocytes and the mechanisms and consequences of decreased F13A1 in RHD. METHODS: We performed studies in platelets, human erythroleukemia (HEL) cells, and human CD34+ cell-derived megakaryocytes including on clot contraction in cells following small inhibitor RNA knockdown (KD) of RUNX1 or F13A1. RESULTS: Platelet F13A1 mRNA and protein were decreased in our index patient and in 2 siblings from an unrelated family with RHD. Platelet-driven clot contraction was decreased in the patient and affected daughter. Promoter studies in HEL cells showed that RUNX1 regulates F13A1 transcription; RUNX1 overexpression increased, and small inhibitor RNA RUNX1 KD reduced F13A1 promoter activity and protein. Following RUNX1 or F13A1 KD, clot contraction by HEL cells was decreased, as were FXIII-A surface expression, myosin light chain phosphorylation, and PAC1 antibody binding upon activation. F13A1 expression and clot contraction were impaired in RUNX1 downregulation in human megakaryocytes. CONCLUSION: RUNX1 regulates platelet-megakaryocyte F13A1 expression, which is decreased in RHD, reflecting regulation of a coagulation protein by a hematopoietic transcription factor. Platelet and megakaryocyte clot contraction is decreased in RHD, related to multiple impaired mechanisms including F13A1 expression, myosin phos- phorylation, and αIIbβ3 activation

HIV-1, miRNAs and neuronal deregulation

11th International Symposium on NeuroVirology, May 29 - June 2, 2012, New York, N

PCTP (Phosphatidylcholine Transfer Protein) is Regulated By RUNX1 in Platelets/Megakaryocytes and is Associated with Adverse Cardiovascular Events

Abstract Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a major hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, platelet function defects, and increased leukemia risk. We have described a patient with multiple abnormalities in platelet aggregation and secretion responses with a heterozygous RUNX1 nonsense mutation (Sun et al Blood 2004; 103; 948-54). Transcript expression profiling of patient platelets (Sun et al J Thromb Haemost 2007; 5:146-54)showed several genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP regulates the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Platelet PCTP expression is associated with increased platelet aggregation and calcium mobilization upon activation of protease-activated receptor 4 (PAR4) thrombin receptors in black subjects as compared to white subjects (Edelstein et al Nat Med 2013; 19:1609-16). Pharmacologic inhibition of PCTP decreased platelet aggregation in response to PAR4 agonist and siRNA knockdown of PCTP in megakaryocytic cells blunted calcium mobilization induced by PAR4 (Edelstein et al Nat Med 2013; 19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets and its role in cardiovascular events. Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1 and contributes to cardiovascular events. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to ~ 1 kb of the PCTP 5' upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding site 1 (-345/-340), site 3 (-632/-627), and encompassing sites 4 and 5 (-974/-969, -997/-992). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-345/-340) and both sites 4 and 5 (-974/-969, -997/-992). PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. To assess the clinical relevance of the findings, the relationship between RUNX1 and PCTP in peripheral blood RNA, and PCTP and death or myocardial infarction (MI) events were assessed in two separate patient cohorts (n = 587 total patients) with cardiovascular disease. RUNX1 is transcribed from two alternate promoters (P1 and P2) resulting in different isoforms. In both patient cohorts, there was strong correlation between RUNX1 and PCTP expression in a promoter specific manner. RUNX1 P1 probe sets were strongly and inversely correlated with PCTP expression (p &lt; 0.0001), while the P2 probe sets were not. PCTP expression was associated with death or MI in both patient cohorts (odds ratio 2.1, 95% CI [1.61-2.95], P-value &lt; 0.0001) independent of age, sex, race, platelet count, and cardiovascular risk factors. Conclusions: Our results provide evidence that PCTP is regulated by RUNX1 (potentially in a promoter specific manner), and that PCTP expression is associated with death or myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events. Disclosures No relevant conflicts of interest to declare. </jats:sec

Circulating Plasma Levels of Connective Tissue Growth Factor (CTGF) Are Elevated In Patients Afflicted with Rheumatoid Arthritis

Abstract Abstract 4320 Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA are at an increased risk to develop cardiovascular diseases and the precise mechanism(s) of action remain obscure. This laboratory has proposed the existence of a pro-inflammatory axis in RA comprised by thrombospondin-1 (TSP1), transforming growth factor-beta (TGF-b) and CTGF. The present study evaluated plasma levels of TSP1, TGFb and CTGF in patients with RA by ELISA as well as specific cytokines and chemokines. CTGF plasma levels in RA patients (9.2 pg/ml mean, range 3.44–17.08) were found significantly increased (P&lt;0.047) when compared to control subjects (mean 4.43 pg/ml, range 1.62–7.35). This is the first report in the medical literature documenting an augmented human plasma circulating levels of CTGF in RA. TSP1 circulating levels were found elevated in RA patients when compared to control subjects (mean 315 ng/ml vs. 25.4 ng/ml respectively, P&lt;0.039). TGFb showed a trend for higher levels in patients with RA (10.1 ng/ml vs. 4.5 ng/m, P&lt;0.095). The source CTGF in plasma could not be determined precisely but there is evidence of platelet activation in RA (Platelets 19:146, 2008), and TSP1 is the major content of the platelet a-granule (25%). CTGF can attract monocytes and both have been found co-localized in atherosclerotic plaques (Circulation 95, 831, 1997). IL-6 (P&lt; 0.05), IL-12p70 (P&lt;0.04), IP-10 (P&lt;0.03) and MIP1b (P&lt;0.016) were increased in the RA patients when compared to controls. Recent findings have placed platelet microparticles (Science 327; 580, 2010) as playing a major role (arsonists) in RA via monocytes and neutrophils. In summary, our results provide evidence that a pro-inflammatory axis is active in RA potentially contributing to cytokine changes as well as the cardiovascular disease associated with RA since TSP1 can induced IL-6 generation from monocytes and can activate inert TGFb on cell surfaces (fibroblasts and synoviocytes) with the subsequent up-regulation of CTGF. Disruption of the axis in experimental models of RA may prove to be an emerging therapeutic target in RA. Elucidation of the source of CTGF should provide as well key information for cell-targeting to prevent CTGF secretion into the blood milieu. Disclosures: No relevant conflicts of interest to declare. </jats:sec