Psoriasis is an immune mediated inflammatory disease which affects 2-3% of the world’s population. Over the last decade, psoriasis has been acknowledged as an independent risk factor for atherosclerosis. The precise mechanism or mechanisms of the heightened risk is widely speculated. Endothelial cells and macrophages are central players in the immunopathological development of both diseases. Interleukin-36 cytokines (IL-36) have been heavily implicated in psoriasis immunopathology. Significant upregulation of epidermal IL-36 is a recognised characteristic of psoriatic skin inflammation. IL-36 induces inflammatory responses in dendritic cells, fibroblasts and epithelial cells. While vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in dermal inflammation, the effects of IL-36 on endothelial cells have not been defined. We report that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated following IL-36γ stimulation, and this is reversed in the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. Both resident and infiltrating inflammatory myeloid cells contribute to the immunopathology of psoriasis by promoting the IL-23/IL-17 axis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23, TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. Furthermore, IL-36γ stimulated macrophages potently activated endothelial cells as illustrated by ICAM-1(CD54) upregulation, and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, monocytes isolated from psoriasis patients showed increased adherence to both the stimulated and unstimulated endothelium when compared to monocytes from healthy individuals. Collectively, these findings add to the growing evidence for IL-36γ having roles in psoriatic responses, by enhancing endothelium directed leukocyte infiltration into the skin and strengthening the IL-23/IL-17 pathway. Our findings also point to a cellular response which could potentially support cardiovascular comorbidities in psoriasis.

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Background: Hyperhomocysteinemia (HHcy) is an established risk factor for thrombotic diseases yet the underlying mechanism remain unclear. In this study we investigated the effect of HHcy on endothelial cell-platelet interaction and its role in thrombosis. Methods and Results: We used a novel mouse model of HHcy (plasma homocysteine, Hcy 80 micromolar) in which a Zn2+ inducible human cystathionine beta-synthase (CBS) transgene was introduced to circumvent the neonatal lethality of the Cbs gene deficiency (Tg-hCBS Cbs-/- mice). Hcy-lowering therapy was performed by giving ZnSO4 water to induce human CBS transgene expression in adult mice. Thrombus formation was examined by photo dye-induced cremaster microvasculature thrombosis using intravital microscopy, in which endothelium was preserved, and by FeCl3-induced carotid artery thrombosis, which denudated the endothelium. HHcy accelerated cremaster arteriolar thrombosis and decreased blood flow cessation time from 41.8 min in control mice to 30.5 min in TghCBS Cbs-/- mice. Venular blood flow cessation time was slightly decreased from 5.6 to 5.0 min. Hcy-lowering therapy reduced Hcy level from 80 micromolar to 6.8 micromolar after 2 weeks of ZnSO4 water and prolonged arteriolar blood cessation time from 30.5 to 37.8 min. Interestingly, FeCl3-induced carotid artery thrombosis did not change the occlusion time. Hcy did not potentiate the aggregation and secretion function in washed human platelets from healthy donor treated with Hcy (50, 100 micromolar) or from Tg-hCBS Cbs-/- mice. However, inter-cellular adhesion molecule 1 (ICAM-1) levels, but not vascular adhesion molecule 1 (VCAM-1), were increased in cremaster tissues from Tg-hCBS Cbs-/- mice by western blot. In cultured human umbilical vein ECs (HUVEC), Hcy (100 micromolar, 24h) promoted human platelet adhesion by 200% in static adhesion assay. Using western blot, FACS and RT-PCR, we found that Hcy increased protein and mRNA levels of ICAM-1, but not that of VCAM-1, in HUVEC. ICAM-1 blocking antibody partially reversed Hcy increased platelets adhesion to HUVEC. Hcy induced ICAM-1 expression and reduced DNA methylation on ICAM-1 promoter, which were mimicked by DNA methyltransferase inhibitor azacytidine, and histone deacetylase inhibitors sodium butyrate and trichostatin A. Hcy treatment also increased intracellular Hcy, Sadenosylhomocysteine (SAH) accumulation and decreased SAM/SAH ratio in HUVECs. Hcy decreased methyl CpG binding protein 2 (MeCP2) binding and increased acetylated histone H3 (AcH3) binding to ICAM-1 core promoter region using chromatin immunoprecipitation. Pyrosequencing of ICAM-1 core promoter and adjacent region shows a decreased DNA methylation by Hcy treatment. In high methionine diet-induce HHcy in WT and Icam-/- mice, Icam-/- mice fed with HM diet only show moderately accelerated venular and barely accelerated arteriolar occlusion time compared with WT mice with CT diet using photo dye-induced thrombosis model. Conclusion: HHcy accelerates arteriolar thrombosis and increases EC-platelet interaction via ICAM-1 induction partially through DNA hypomethylation. / Pharmacology

It is well established that cytosolic caspase-1 activation, mediated by inflammasome after pathogens-associated molecular patterns (PAMP) and metabolic danger-associated molecular patterns (DAMPs), mediates the initiation of inflammation in endothelial cells by its downstream targets such as Interleukin-1β (IL-1β), Interleukin-18 (IL-18), and Sirtuin-1. However, it remains unknown whether proatherogenic lipids lysophosphatidylcholine (LPC) and reactive oxygen species (ROS) can promote nuclear localization of caspase-1. Using biochemical, bioinformatic, and immunologic approaches, we made the following findings: (1) DNA damage was found in atherosclerotic mice. (2) A nuclear exportation signal was mapped in the CARD domain of pro-caspase-1. LPC promotes nuclear localization of pro-caspase-1 in human aortic endothelial cells (HAECs), which may interrupt DNA damage and repair pathways. (3) Blockage of caspase-1 nuclear cytosol trafficking in HAEC activated by LPC may mediate inflammation and interrupt cell cycle regulation. (4) Pro-caspase-1 in the nucleus inhibits inflammation but promotes interferon pathways. Activation of caspase-1 in the nucleus promotes aging- and fos-related antigen 2 (FRA2) mediated DNA damage and apoptosis. (5) Inhibition of SUMOylation decreases pro-caspase-1 translocation into the cytosol from the nucleus. (6) Blockage of caspase-1 cytosol nuclear trafficking in HAEC activated by H2O2 may decrease caspase-1 activity and increase cell viability. Our results demonstrate, for the first time, that caspase-1 patrols in the cell, senses danger signals and interrupts the balance between DNA damage and DNA repair pathways. It is a novel insight that not only should we suppress the inflammation in the cytosol but also in the nucleus, which is important for the future development of therapeutics for cardiovascular diseases and other inflammatory diseases. / Biomedical Sciences

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD). We previously demonstrated that homocysteine (Hcy) suppresses endothelial cell (EC) proliferation, migration, and post-injury EC repair, but the molecular mechanism underlying Hcy-induced EC injury is unclear. In this study, we identified a novel gene, Carom, which mediates Hcy-induced suppression of EC migration and angiogenesis. We identified FCH and double SH3 domains 2 (FCHSD2), a novel gene, as an Hcy-responsive gene through Differential Display in Hcy (50µM)-treated human umbilical vein endothelial cells (HUVEC). FCHSD2 was initially named as Carom, based on the identification of this molecule as an interacting protein of calcium/calmodulin-dependent serine protein kinase (CASK) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1). In this thesis, we describe this gene as Carom. Carom belongs to the Fes/CIP4 homology and Bin/amphiphysin/Rvs (F-BAR) protein family, which is a group of multivalent adaptors linking plasma membrane and cytoskeleton, involved in endocytosis and cell migration. However, Carom's function is poorly characterized. Based on the findings that CASK and MAGI1 inhibit cell migration and growth, and the role of F-BAR proteins in cell migration, we hypothesize that Hcy up-regulates Carom to inhibit EC growth and/or migration, finally leading to CVD. We confirmed the significant induction of Carom mRNA expression in Hcy-treated HUVECs or human aortic endothelial cells (HAEC) by Northern blot and Real-time PCR. In addition, we found that Carom protein expressions were significantly increased both in Hcy-treated HAECs and lung ECs isolated from HHcy mice by Western blot using our homemade rabbit antibody against Carom. These data indicate that Hcy increases endothelial expression of Carom both in vitro and in vivo. Furthermore, in order to characterize Carom function in EC, we generated recombinant adenovirus Adv-Carom to transduce Carom for gain-of-function study and Adv-Carom-shRNA to express Carom shRNA for loss-of-function study. We found that neither adenovirus-transduced Carom expression nor adenoviral Carom shRNA had any impact on HUVEC proliferation by using [3H]-thymidine incorporation. Interestingly, we demonstrated that Adv-Carom inhibited HAEC migration, while Hcy-induced HEAC migration inhibition could be rescued by Adv-Carom-shRNA. These data suggest that Carom may inhibit angiogenesis via a cell proliferation-independent mechanism. Furthermore, we found that Hcy significantly increased the intracellular level of S-adenosyl homocysteine (SAH) but not S-adenosyl methionine (SAM), and decreased the SAM/SAH ratio, an indicator of cellular methylation, in HAECs, by using High-performance liquid chromatography/electrospray tandem mass spectrometry (HPLC-MS) to measure SAH and SAM levels. Meanwhile, Carom protein expression was significantly induced by azacytidine (AZC), a DNA methyltransferse inhibitor, in a dose-dependent manner in HAECs. Based on these data, we speculated that Hcy-induced hypomethylation could associate with Carom up-regulation. Thus we used bisulfite deep sequencing to profile methylation status of Carom gene in Hcy-treated HUVECs and found that Carom promoter was hypomethylated by Hcy. In addition, eight transcriptional factor binding sites on Carom were hypomethylated by Hcy. These data suggest that Hcy may induce Carom via a DNA hypomethylation-dependent mechanism. Moreover, we found that adenovirus-transduced Carom expression significantly increased the secretions of two anti-angiogenic chemokines, CXCL10 and CXCL11 in HAECs by using human cytokine array. Similarly, Hcy also significantly increased mRNA expressions of CXCL10 and CXCL11, while Adv-Carom-shRNA blocked down the inductions of CXCL10 and CXCL11 by Hcy. We further demonstrated that adenovirus-transduced Carom expression inhibited angiogenesis by performing tube formation assay of HAECs, whereas Hcy-induced angiogenesis suppression were rescued by Adv-Carom-shRNA as well as the neutralizing antibodies of CXCL10 and CXCL11. These data suggest that Hcy induces Carom to trigger CXCL10 and CXCL11 downstream to inhibit angiogenesis. In conclusion, Hcy induces Carom expression through DNA hypomethylation to inhibit EC migration and angiogenesis. / Pharmacology

Ascorbate (Asc) is a key antioxidant in preventing cardiovascular dysfunction during diseases exacerbated by altered shear stress. According to the literature endothelial responses to hypotonic shock share some characteristics with those induced by shear stress. Thus to study the physiological responses of endothelium to shear stress, the characterization of the Asc release by pig coronary artery endothelial cells in response to hypotonic shock was performed. The pig coronary artery endothelial cells that had been loaded with ^14C Asc and ^3H deoxyglucose, were exposed to buffers of varying osmolality for different time periods and the release of ^14C Asc and ^3H deoxyglucose was examined. Based on various parameters like relative release of ^14C Asc and ^3H deoxyglucose, their rate of release and protein loss, it was decided to use buffer of .67 percent osmolality for 2 min for these characterization studies. The Asc release was authentic and not a result of membrane damage. The hypotonic shock induced Asc release was not due to endogenously released ATP. The inhibition of ATP induced release by anion channel inhibitors niflumic acid and NPPB was complete but only partial in case of hypotonic shock induced release. The release was not inhibited under nominally Ca^2+ free conditions. Additive release by hypotonic shock and ATP or hypotonic shock and Ca^2+ ionophore A23187 suggests that there are two independent Asc release pathways. Asc release by two different mechanisms may help endothelial cells deal with stressful conditions efficiently and preserve endothelial function. / Thesis / Master of Science (MS)

Blood vessels are critical for the delivery of oxygen and nutrients to all cells in the body. To properly function, blood vessels and their primary components must develop and mature into a healthy network, capable of dynamic alterations to meet new needs of the body. The early genetic and molecular programs that "push" the vasculature to develop are the same programs that reactivate when there are normal changes to the body such as injury, muscle growth or decline, or aging; and when pathologies arise like cancer, stroke, and diabetes. Therefore, it is crucial to understand how the vasculature develops into a healthy system by studying all components as they mature. Endothelial cells that comprise the vessels themselves are joined by specialized partner cells called pericytes that help guide and mature vessel growth. Pericytes lie elongated along endothelial cells and have multiple points of contact with the endothelium. In this position, pericytes assist in cell-cell communication and even blood flow regulation in the microvasculature. To study the relationship between endothelial cells and pericytes during development, we observed vascular morphology in three and four dimensions, as well as the genetic and molecular mechanisms underlying how these cells are recruited and interact in several experimental models. Thus, to thoroughly analyze the morphology of these vessels, we developed a rigorous methodology using a MATLAB program to determine the colocalization and coverage of pericytes associated with vessels in large image sets. After developing analytical methods to investigate all the components of the blood vessel wall, we expanded our investigation of how pericytes and other aspects of microvasculature develop in animal models, specifically a more commonly used murine model for vascular development and for treatment of human diseases. Our findings of vascular development in mice suggest that there are important differences in how human and mouse brain blood vessels form. Therefore, studies using mice must be carefully designed to account for these discrepancies. Additionally, research into why human and mouse neurovascular development and maturation are different can aid in the development of improved experimental models to better treat human pathologies. / Doctor of Philosophy / Blood vessels have the crucial job of delivering oxygen and nutrients to all the cells in the body. To perform this duty, blood vessels- and the components that make them- must develop and mature into a healthy network, capable of altering itself to meet new needs of the body. The early programs that “push” the vessel system to develop are the same programs that reactivate when there are normal changes to the body such as injury, muscle growth or decline, or aging; and when abnormal diseases arise like cancer, stroke, and diabetes. Therefore, it is critical to understand how blood vessels develop into healthy systems by studying all of their components as they mature. Endothelial cells that comprise the vessels themselves are joined by specialized partner cells called pericytes that help guide and mature vessel growth. Pericytes lie elongated along endothelial cells and have multiple points of contact with the endothelium. In this position, pericytes assist in cell-cell communication and even blood flow regulation in smaller vessels called capillaries. To study the relationship between endothelial cells and pericytes during development, we observed vascular anatomy in three and four dimensions, as well as mechanisms underlying how these cells come together and interact in several experimental models. Thus, to thoroughly analyze the morphology of these vessels, we developed a rigorous methodology using a MATLAB program to determine the colocalization and coverage of pericytes associated with vessels in large image sets. After developing analytical method to investigate all the components of the blood vessel wall, we expanded our investigation of how pericytes and other aspects of blood vessels develop in animal models, specifically a more commonly used animal model for vascular development and for treatment of human diseases. Our findings of vascular development in mice suggest that there are important differences in how human and mouse brain blood vessels form. Therefore, studies using mice must be carefully designed to account for these discrepancies. Additionally, research into why human and mouse neurovascular development and maturation are different can aid in the development of improved experimental models to better treat human illness and injury.

No / Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-B by measuring the loss of its inhibitor I-B. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-B. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-B activation.