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Identifier

000374489

Title

Γενετική ανάλυση της αγγειογένεσης στο συστηματικό ερυθηματώδη λύκο : γονιδιακοί πολυμορφισμοί σε ειδικά μόρια του ενδοθηλίου και η συμβολή τους στη μελέτη της ενδοθηλιακής βιολογίας και της αγγειογένεσης

Alternative Title

Genetic analysis of angiogenesis in systemic lupus erythematosus :gene polymorhisms in endothelial cell specific molecules and their implications for the endothelial biology and angiogenesis

Author

Βαζγιουράκης, Βασίλειος Μ

Thesis advisor

Μπούμπας, Δημήτριος

Reviewer

Γουλιέλμος, Γεώργιος
Σιδηρόπουλος, Πρόδρομος
Καρδάσης, Δημήτριος
Παπαδάκη, Ελένη
Ηλιόπουλος, Αριστείδης
Τσατσάνης, Χ,

Abstract

The immune system has evolved to protect the body from foreign invading pathogens. Unfortunately under certain conditions the immune tolerance is lost and the immune system attacks the host causing damage in several tissues and organs, situation that is observed in various autoimmune diseases. Systemic Lupus Erythematosus (SLE) is the prototype autoimmune disorder that is principally characterized by hyper activated B-cells, autoantibodies production and aberrant activation of the immune system leading to chronic inflammation. Among the various systems affected in SLE is the vascular system and cardiovascular disorders account for increased morbidity and mortality during the course of the disease. SLE patients are characterized by accelerated and premature atherosclerosis that can’t be fully explained by the traditional Framingham risk factors. Several non-traditional risk factors have been proposed such as systemic inflammation that impairs endothelial cell function and autoantibodies against endothelial cell constituents that cause damage and cell death. Further attack of the cellular immunity to endothelial cells and inappropriate endothelial repair leads ultimately in endothelial dysfunction that can be recognized both with clinical methods and biological-cellular markers such as the increased number of CECs and decreased EPCs numbers in the blood of SLE patients. Atherosclerosis is now considered as an inflammatory and autoimmune disease. Many constituents of both innate and adaptive immunity have been recognized in atherosclerotic plaques and may account for the pathogenesis of the disease. The initiating event is the endothelial dysfunction followed by endothelial layer denudation and further progression due to aberrant angiogenesis that takes place inside the evolving plaque. Although several mechanisms have been proposed, the exact nature of excessive vasculopathy in SLE remains elusive. Signaling pathways in endothelial cells that can be activated or influenced by the immune system may be excellent candidates for further studies. CD40 is expressed in several cell types including Β-lymphocytes, monocytes/macrophages and endothelial cells and is considered of outmost importance for efficient humoral immune responses. On the other hand CD40 signalling exerts proinflammatory functions in endothelial cells and monocytes, accelerates atherosclerosis and induces aberrant angiogenic responses due to inappropriate production of VEGF. Although CD40 has recently been identified in a genome-wide association study as a novel rheumatoid arthritis susceptibility gene such an association has not been documented for SLE. Nevertheless, sustained CD40L expression by T cells and platelets in SLE activates a variety of cells via its receptor CD40 contributing to disease pathogenesis and increased levels of CD40L have been documented in plasma of SLE patients. VEGF is considered to be the principal angiogenic growth factor that mediates signals for endothelial cell proliferation and viability and induces endothelial cell migration and sprouting, thus contributing to efficient angiogenesis. VEGF is produced by endothelial cells, monocytes, fibroblasts and SMCs in response to hypoxia which is the major physiological signal for angiogenesis. However, under certain pathological conditions, VEGF can be produced by other cell types such as tumor cells or activated T-lymphocytes inducing inflammatory angiogenesis, known as neo-angiogenesis. Neo-vessels are immature vessels, with no or little mural cells, fragile and leaky. Such vessels are abundant in atherosclerotic plaques and are considered as one of the major factors for intraplaque hemorrhage and instability leading to vulnerable atherosclerotic plaques. VEGFR2 is the principal receptor that mediates VEGF signals in endothelial cells and belongs to the receptor-tyrosine kinases superfamily. The extracellular region of the receptor is composed of 7 Ig-like domains with the ligand binding region located on the 2nd-3d Ig-like domains. The 4th-7th Ig-like extracellular domains are considered crucial for signal transduction. Upon ligand binding, the receptor dimerizes and autophosphorylated transmitting signals to other intracellular kinases. VEGFR2 has been associated with vascular diseases such as coronary artery disease and genetic alterations that influence its signaling have been found in certain vascular tumors but its implication in SLE has not been investigated. Taken together, the endothelial expression of both CD40 and VEGFR2, their ascribed role in immune regulation and endothelial function and the possible cross-talk between their signaling cascades sought us to explore possible associations of SNPs in these two molecules with SLE thus implicating them in disease pathogenesis. For genetic association study, the primary sample set consisted of 351 SLE patients and 670 matched healthy controls of Greek origin. We used 158 SLE patients and 155 controls from Turkey as replication sample. Genotyping of rs4810485 (that has been associated with rheumatoid arthritis) was performed by RFLPs and the Sequenom MassArray technology. The expression of CD40 mRNA and protein was assessed in unstimulated and LPS-stimulated peripheral blood mononuclear cells by quantitative real time PCR and flow cytometry, respectively. CD45 negative cells were isolated from peripheral blood and the expression of CD40 in CECs subfraction of these cells was also assessed by flow-cytometry. The V297I (rs2305948) and Q472H (rs1870377) single nucleotide polymorphisms (SNPs) in VEGFR2 located in third and fifth extracellular IgM-like domains were genotyped with Taqman technology and RFLPs in 150 SLE patients and 96 healthy controls. CECs were isolated from whole blood from 54 SLE patients with CD146-coated magnetic beads and enumerated after staining with TRITC-labeled Ulex Europaeus Aglutinin-1 according to standard protocols. Modeling of the mutation V297I was based on the 3D structure of domains D2 and D3 of VEGFR2 in complex with VEGF-C. Sequence alignment of the D5 domain of VEGFR2 was performed on the KIT sequence, followed by homology modeling on the KIT ectodomain structure to investigate mutation Q472H. The minor allele T of CD40 rs4810485 SNP was significantly under-represented in Greek SLE patients compared to healthy controls. The association was replicated in the Turkish cohort and remained statistically significant after the meta-analysis of 509 SLE patients and 825 healthy controls. In both cases and controls, the rs4810485 G/T and T/T genotypes were associated with significantly reduced CD40 mRNA and protein expression in peripheral blood CD14+ monocytes and CD19+ B cells compared to G/G genotype, both under basal conditions and following antigen stimulation. G/T and T/T genotypes were also associated with reduced numbers of total and CD40+ CECs and reduced fluorescence intensity of CD40 on CD40+ CECs. The risk allele A of the VEGFR2 rs1870377 SNP was more frequent in individuals with SLE than in healthy controls, while A/A genotype appeared to be a SLE risk factor. No association was detected between rs2305948 and SLE. Within the rs1870377 A/A genotype the number of CECs was significantly higher than that of T/T genotype. Modeling revealed that amino acid position #472 which is located on the D3 D5 Ig-like domain of the extracellular region of VEGFR2 affects the efficiency of trans-autophosphorylation and possibly the receptor activation and cell signaling. In conclusion we have identified CD40 as a novel susceptibility locus in Greek and Turkish SLE patients. The rs4810485 minor allele T is under-represented in SLE and correlates with reduced CD40 expression in peripheral blood monocytes, B cells and endothelial cells. This fact indicates that majority of SLE patients is characterized by higher CD40 expressions both in immune and endothelial cells and higher numbers of CECs indicative of increased vascular damage. These findings may account for increased CD40 signaling in immune and endothelial cells and may have implications both in the regulation of aberrant immune responses and increased endothelial cell activation and damage due to increased CD40 signaling. The VEGFR2 Q472H polymorphism is associated with increased susceptibility to SLE, thus implicating VEGFR2 in pathogenesis of the disease. The risk genotype for rs1870377 SNP was associated with higher CECs numbers, indicating that this mutation may correlate with increased endothelial damage. Structural data suggest that this mutation may cause impairment in cell signaling, thus contributing to SLE pathogenesis. Our overall findings identified CD40 and VEGFR2 genes as candidates in SLE pathogenesis and provide evidence to support the hypothesis that their pathways may implicated in endothelial biology and angiogenesis in SLE. It’s intriguing to speculate that in addition to aberrant immune responses, CD40 signaling in B-cells and monocytes mediates aberrant autoantibody production and endothelial cell damage along with the overproduction of VEGF. Increased VEGF binding to VEGFR2 that transmits impaired signals may further compromise endothelial integrity. This mechanism can serve as working hypothesis that of course needs prove experimental validation in order to be proved.

Language

Greek

Subject

Angiogenesis

Atherosclerosis

Autoimmunity

Endothelium

Immune mechanisms

Inflammation

Lupus Erythematosus, Systemic

Molecular genetics

Systemic lupus erythematosus