Anti-Beta2-Glycoprotein I antibodies (anti-b2GPI) are strongly associated with thrombosis in patients with primary antiphospholipid syndrome (PAPS). Anti-b2GPI activate endothelial cells (EC) resulting in a pro-thrombotic and pro-inflammatory phenotype. In order to characterise EC gene regulation in response to anti-b2GPI, early global gene expression was assessed in human umbilical vein endothelial cells (HUVEC) in response to affinity purified anti-b2GPI. Sera were collected from patients with PAPS and IgG was purified using HiTrap Protein G Sepharose columns. Polyclonal anti-b2GPI were prepared by passing patient IgG through NHS activated sepharose coupled to human b2GPI. Anti-b2GPI preparations were characterized by confirming their b2GPI co-factor dependence, binding to b2GPI and ability to induce leukocyte adhesion molecule expression and IL-8 production in vitro. Two microarray experiments tested differential global gene expression in 6 individual HUVEC donors in response to 5 different PAPS polyclonal anti-b2GPI (50 mg/ml) compared to 5 normal control IgG (50 mg/ml) after 4 hours incubation . Total HUVEC RNA was extracted and cRNA was prepared and hybridised to Affymetrix HG-133A (Exp.1) and HG-133A_2 (Exp.2) gene chips. Data were analyzed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. Significant change in gene expression was defined as greater than two fold increase or decrease in expression (p<0.05). Novel genes not previously associated with PAPS were induced including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1 and growth factors, CSF2, CSF3, IL-6, IL1b and FGF18. Downregulated genes were transcription factors/signaling molecules including ID2. Microarray results were confirmed for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C) using quantitative real-time RT-PCR analysis. This study revealed a complex anti-b2GPI-regulated gene expression profile in HUVEC in vitro. The novel chemokines and pro-inflammatory cytokines identified in this study may contribute to the vasculopathy associated with PAPS.

Darbo tikslas: Nustatyti adenozino receptorių (AR) genų raišką (informacinės RNR lygyje) plaučių kraujagyslių endotelio ląstelėse ir žiurkės plaučiuose, jų pokyčius esant hipoksijai, įvertinti AR poveikį endotelio ląstelių proliferacijai. Darbo uždaviniai: 1) Ištirti žiurkės plaučių smulkių kraujagyslių endotelio (PSKE) ir žiurkės plaučių arterijos endotelio (PAE) AR genų raišką, veikiant hipoksijai. 2) Nustatyti AR agonistų poveikį PSKE ląstelių proliferacijai. 3) Nustatyti AR genų raiškos pokyčius žiurkės plaučiuose esant hipoksijai. 4) Nustatyti adenozino receptorių A3 (A3R) pasiskirstymą plaučių arterinės hipertenzijos (PAH) paciento plaučių mėginyje, taikant imunohistocheminį tyrimą. Darbo metodai: genų raiška nustatyta taikant kiekybinę tikro laiko polimerazės grandininę reakciją (KTL-PGR) naudojant “Taqman®” pradmenis ir zondus adenozino receptoriams. Ląstelių proliferacija įvertinta tričiu žymėto timidino (3H-timidino) įjungimu į ląstelių DNR. A3R pasiskirstymas PAH paciento plaučiuose įvertinamas, taikant imunohistocheminį tyrimą. Tyrimo rezultatai: PSKE ląstelėse rasta adenozino receptorių A2B ir A3R. Hipoksijos aplinkoje A2BR genų raiška padidėjo 2 kartus po 24 val., 5 kartus po 40 val. A3R genų raiška sumažėjo 2 kartus po 24 val., 6 kartus po 40 valandų. PAE ląstelėse rasta AR: A1, A2AR ir A2BR. Hipoksijos aplinkoje A1R genų raiška padidėjo 2,5 karto po 24 val. ir išliko padidėjusi po 40 val. A2AR genų raiška padidėjo 2 kartus po 24 val. ir išliko padidėjusi... [toliau žr. visą tekstą] / Aims: Determine adenosine receptors (AR) gene expression in rat pulmonary microvascular endothelial cells (RPMVEC), rat pulmonary artery endothelial cells (RPAEC) and rat lungs during hypoxic conditions. Evaluate effects of adenosine receptors agonist to PRMVEC proliferation. Objectives: 1) Identify AR in RPMVEC and RPAEC. 2) Evaluate AR changes in RPMVEC and RPAEC during hypoxic conditions. 3) Determine adenosine receptors expression in rat lungs exposed to chronic hypoxia. 4) Perform immunohistochemical staining of A3R on a lung section from patient with pulmonary arterial hypertension (PAH). Methods: Adenosine receptors gene expression was determined by quantitative real time polymerase chain reaction (qRT-PCR) assay using “TaqMan®” primers. Cell proliferation was determined using a tritium labeled thymidine (3H-thymidine) assay. Immunohistochemistry was performed on paraffin embedded lung tissue sections. Results: RPMVEC express A2BR and A3R. During hypoxic conditions A2BR was upregulated 2-fold after 24 h. and 5-fold after 40h of hypoxic exposure. A3R was downregulated 2- fold after 24h. and 6-fold after 40h. RPAEC express A1R, A2AR and A2BR. During hypoxic conditions A1R expression was increased 2,5-fold after 24h and 40h. A2AR was upregulated 2-fold after 24h and 40h. A2BR expression was increased 2,5-fold after 24h and 40h of hypoxic exposure. The A3R agonist HEMADO treatment for 24h at the concentration of 10-7 M, increased RPMVEC proliferation 1,5-fold. AR... [to full text]

Atherosclerosis is an inflammatory disease, occurring preferentially in arterial regions with disturbed flow. We have shown that disturbed flow induces inflammation in endothelial cells (ECs) by producing bone morphogenic protein-4 (BMP4). Moreover, chronic BMP4 infusion induces endothelial dysfunction and systemic hypertension in mice. Here, we examined which BMP receptors (BMPR) mediate BMP4 action in ECs. Western blot, immunostaining and RT-PCR studies using human and bovine ECs, mouse aortas and human coronary arteries (HCA) showed that BMPRI (ALK2 and 6) and BMP-RII were expressed in ECs. As a functional test, ECs were treated with a BMPRII siRNA to knockdown expression. BMPRII knockdown blocked a well-known BMP4 response - smad1/5/8 phosphorylation, as expected. Unexpectedly, BMPRII knockdown itself significantly stimulated ICAM-1 and VCAM-1 expression and monocyte adhesion in a BMP4-independent manner. Inflammatory responses caused by BMPRII knockdown were blocked by inhibitors of NADPH oxidase and NFκ B. From these results, we hypothesized that BMP-RII knockdown in ECs would cause inflammation, which is a critical event in atherosclerosis initiation and progression. Genetic mutations of BMPRII have been linked to primary pulmonary hypertension. However, it is not known whether BMP-RII is regulated by atherosclerotic conditions and plays a role in non-pulmonary vessels causing inflammation and atherosclerosis. We examined BMPRII levels in HCA by immunostaining. While non-diseased arteries showed intense staining of BMPRII, the expression decreased as lesions became more advanced. BMPRII was virtually undetectable in the most advanced lesions. These findings suggested a potential link between pro-atherosclerotic conditions and BMP-RII levels. We tested this hypothesis by treating ECs with pro-inflammatory cytokines found in atheromas: TNFα decreased BMPRII by 2-fold. In contrast, statins increased BMPRII by 4-fold. In summary, we demonstrate for the first time that BMPRII can be down- or up-regulated by pro- or anti-atherogenic conditions, respectively, and it is dramatically decreased in HCA with advanced plaques. Moreover, BMPRII knockdown in ECs induces inflammation, a critical atherogenic step. We propose that focal inflammation initiated by disturbed flow, together with circulating pro-atherogenic risk factors, may lead to a vicious cycle of BMPRII down-regulation causing secondary inflammation and atheroma progression.

Doctor of Philosophy (Medicine) / Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 102-118).

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 130-144). Also available online.

Thesis (Ph. D.)--Cornell University, May, 2009. / Vita. Includes bibliographical references (leaves 128-147).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 102-123).

At the present study we examined the effect of fluid shear stress on two different cell types. The cells studied were the Human Umbilical Vein endothelial cells and Smooth Muscle cells. For that purpose, a device which was simulating the arterial circulation was used. Shear stress is the hemodynamic force of blood. We show that this mechanical stress can efficiently parallelize the cellular morphology and induce changes at a gene transcription level. Specifically, we proove that shear stress is responsible for the upregualation of specific endothelial markers whereas can mediate the downregulation of smooth muscle cells markers in both cell types examined. / Στην παρούσα εργασία μελετήθηκε η επίδραση της διατμητικής τάσης ρευστού επάνω σε δυο διαφορετικούς τύπους κυττάρων. Τα κύτταρα που μελετήθηκαν ήταν τα Ανθρώπινα Ενδοθηλιακά κύτταρα απο φλέβα του Ομφάλιου λώρου και τα Λεία Μυικά κύτταρα. Χρησιμοποίηθηκε μια συσκευή η οποία προσομοίωνε την αρτηριακή κυκλοφορία του αίματος. Η διατμητική τάση ρευστού είναι η αιμοδυναμική δύναμη του αίματος. Στην εργασία δείχτηκε πως η δύναμη αυτή μεταβάλει τη μορφολογία των κυττάρων παραλληλίζοντας τα με τη ροή ενώ αυξάνει τα ενδοθηλιακά γονιδία και μειώνει τα λεία μυικά γονίδια και στους δυο τύπους κυττάρων που εξετάστηκαν.