Vijay K. Kalra - US grants

Vasoocclusive crisis is the major cause of morbidity and mortality in sickle cell disease (SCD). The molecular cascade giving rise to slowed blood-vessel flow remains elusive. Slowing of blood flow and hence oxygen delivery to tissues very likely relates to the adherence of sickle cell (SS) RBC to vascular endothelium, localized tissue hypoxia secondary to blood stasis and/or inflammation common in SCD. SS RBC adherence may affect blood flow by promoting, via surface expression of counter- receptors on the endothelium, the additional binding of SS RBC and leukocytes. We hypothesize that SS RBC adherence to endothelium generates cellular oxidant stress (evidenced by lipid peroxide formation and activation of transcription factor NF-kb), leading to a surface expression (SE) of a subset of cell adhesion molecules (CAMs), ICAM-1, E- selectin and BCAM-1. Similarly, infection/inflammation causes an increase in CAM expression. The induced SE of VCAM-1 leads to a greater adherence of less dense SS RBC through their alpha4beta1 ligand. The adherence and subsequent diapedesis of PMN and monocytes occurs via the increased SE of CAMs, which act as counter-receptors for integrins on these cells. Because the activation of NF-kB occurs partly through lipid peroxides generated by oxidant stresses such as vasoocclusion, e plan to determine whether overexpression of bcl-2 (a lipid peroxide-suppressing protooncogene) in cultured human umbilical vein endothelial cells (HUVEC) will prevent lipid peroxidation, NF-Kb activation and a CAM induction when transfected HUVEC are incubated with SS RBC. Dense deoxygenated SS RBC display phosphatidylserine (PS) on the external leaflet of the RBC bilayer and adhere to HUVEC through the PS receptor, so we will determine whether LPS and cytokines, which induce the expression of PS receptor, potentiate dense deoxygenated SS RBC adherence. Additionally, hypoxia increases monocyte trans endothelial migration, which may contribute to vasoocclusion; thus we propose investigating the mechanism of hypoxia-induced diapedesis of PMN and monocytes. We will also examine the mechanism(s) by which SS RBC, LPS and cytokine interaction with HUVEC alters CAM expression, the concomitant adherence of PMN and monocytes, and the augmented SS RBC adherence. Moreover, we will investigate CAM induction in endothelial cells derived from distinct vascular beds (pulmonary microvessel, pulmonary artery and brain), and potential differences in such induction effected by various shear stresses, mimicking conditions prevalent in various vascular beds. These completed studies will provide insight into new pharmacological and/or molecular approaches to ameliorate the clinical manifestations of vasoocclusion in sickle cell disease.

Vascular deposition of amyloid-beta-peptide (Abeta) occurs during normal aging and is accelerated in Alzheimer's Disease (AD) and related disorders such as hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D), a form of cerebral amyloid angiopathy (CAA). In CAA, HCHWA-D and AD increased vascular deposition of Abeta is associated with invasion of monocyte/macrophages in the vessel wall and activated microglial cells in the parenchyma. Relatively little is known about the cellular and molecular mechanisms in cerebral vasculature that may play a role in Abeta-mediated accumulation of peripheral inflammatory cells. Recent studies indicated that peripheral hematopoietic cells (i.e., monocytes) can cross the blood-brain barrier (BBB). Our studies shown that Abeta increases diapedesis of leukocytes across the vascular endothelium, and induces migration of monocytes across the human BBB in vitro, blocked by antibodies to PECAM-1 ( a cell junction molecule ) and RAGE (putative receptor for A beta). The central hypothesis is that interaction of Abeta with its putative receptor(s) in the brain vascular endothelium brings about cellular signaling which results in the modulation of PECAM-1 to allow migration of monocytes across the BBB. It is further hypothesized, that migration of monocytes is exaggerated across cerebral vasculature in AD and aged non-human primates, presumably due to increased expression and/or redistribution of Abeta receptors at the BBB. To test this hypothesis we will determine (1) the effect of Abeta on adhesion and migration of monocytes across monolayer of human brain microvascular endothelial cells (HBMVEC); (2) whether Abeta causes increased migration of monocytes in HBMVEC from geriatric and AD cases; (3) the role of p21/ras, MAP kinase, NF-kappaB,, RAGE and PAF in Abeta induced cellular signaling, using pharmacological inhibitors and over-expressing or knocking out genes by transfection of HBMVEC, and (4) the mechanisms of Abeta-induced adhesion and diapedesis of leukocytes across the vessel wall in transgenic (Tg) CC HCHWA-D mice and Tg vascular RAGE mice. Proposed studies will provide a novel insight into the mechanisms of Abeta-induced migration of hematopoietic cells across the vessel wall and the BB and will provide a molecular basis for the therapeutic rationale to modify Abeta- induced inflammatory reactions in the aging brain to ameliorate the neuronal injury.

DESCRIPTION (provided by applicant): Pulmonary hypertension (PHT) occurs in ~30% of patients with sickle cell anemia (SCA) and results in ~50% mortality within 2 years of diagnosis. The pathogenesis of this vasculopathy is likely multi-factorial, potentiated by hemolysis-induced impaired nitric oxide bioavailability, chronic thrombo-embolism from a procoagulant state, and increased endothelin-1 (ET-1). Our studies have shown that placenta growth factor (PlGF), an angiogenic growth factor produced in high amounts by sickle erythroid cells, induces expression of the potent pulmonary vasoconstrictor ET-1, and a procoagulant, plasminogen activator inhibitor-1 (PAI-1), from human pulmonary microvascular endothelial cells (HPMVEC). PlGF increases ET-1 and PAI-1 expression via induction of hypoxia-inducible factor-11 (HIF-11). PHT can be induced experimentally by ectopic PlGF expression in normal mice characterized by increased ET-1, as is observed in transgenic sickle mice and in SCA patients. We recently observed that PlGF-mediated induction of HIF-11 and PAI-1 in HPMVEC is post- transcriptionally regulated by three specific microRNAs (miRs). Relatively little is known of the post- transcriptional, miR-mediated, regulated expression of HIF-1a, ET-1 and PAI-1, or of the RNA-binding proteins that stabilize the mRNAs of these genes that promote PHT. Thus our overall hypothesis is that cytoplasmic RNA-binding proteins and miRNAs alter the stability of HIF-11, ET-1, and PAI-1 mRNAs, and are directly involved in the development of PHT. To address this hypothesis, in Aim 1, we will determine the post- transcriptional mechanisms which regulate PlGF-mediated expression of HIF-1a, ET-1 and PAI-1 and identify RNA binding proteins and the specific miRs involved in binding to mRNA 3'UTRs thus regulating translation of HIF-1a, ET-1, and PAI-1 mRNAs. In Aim 2, we will determine whether the genes for miRs that regulate PAI-1 expression, and are located within introns of NFYC and SKA2 genes are co-synthesized from the NFYC and SKA2 primary transcripts, or are independently transcribed from a smaller, pre-miRNA transcription unit. In Aim 3, we will demonstrate the requirement of these miRs in the regulation of HIF-11, ET-1 and PAI-1 in genetic mouse models that over-express PlGF and develop PHT. Finally, we will determine the association of plasma levels of these miRNAs to plasma PlGF, ET-1 and PAI-1 in SCA patients with and without PHT symptoms. These studies will advance our knowledge as to how RNA binding proteins and miRs regulate some of the key genes involved in sickle PHT, and how expression of these miRNAs is itself regulated. These studies will likely provide new diagnostic bio-markers for assessment of PHT, and novel therapeutic targets for a disease that currently has few or no therapeutic options. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension (PHT) is associated with high death rate among sickle cell anemia patients. We have found that genes (e.g. endothelin-1) associated with PHT are increased in these patients, and we propose to study specific microRNAs involved in their regulation.