This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

The growth and metastasis of solid tumors are dependent on angiogenesis. An endogenous angiogenesis inhibitor, vasostatin, is the proteolytic fragment derived from the N-terminal 180 residues of calreticulin. Previous studies indicated that vasostatin specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth. However, continuous administration of vasostatin is difficult and expensive to facilitate, thereby underscoring the need to develop gene delivery approach. Adenovirus vectors possess advantages for gene delivery including high titer, high infection efficiency and broad host range. The aim of the present study was to generate and characterize recombinant adenovirus vectors encoding vasostatin (Ad-VS) or Igk-fused vasostatin (Ad-Igk-VS), thereby to evaluate the efficacy of anti-angiogenesis gene therapy for tumor suppression. Recombinant Ad-VS and Ad-Igk-VS were generated and verified by PCR and western blot analysis. In addition, adenovirus encoding angiostatin was also produced as positive control for angiogenesis assays. Adenovirus-mediated vasostatin gene delivery specifically inhibited the proliferation of bovine aortic endothelial cells (BAEC), but not non-endothelial cells such as Hela or NIH3T3 cells. Moreover, vasostatin gene delivery potently inhibited the proliferation, migration and tube formation, but not secretion of matrix metalloproteinases (MMPs), in endothelial cells. Flow cytometry analysis indicated that vasostatin gene delivery induced apoptosis in BAEC. Using western blot analysis, it was revealed that gene delivery of vasostatin increased the levels of Fas and FADD in BAEC. In conclusion, adenovirus-mediated vasostatin gene delivery inhibited various angiogenesis processes at least via induction of Fas/FasL pathway and may hold potential for cancer therapy.

Numerous studies have identified that elevated plasma concentrations of lipoprotein(a) [Lp(a)] are an emerging risk factor for a variety of atherothrombotic disorders. Apolipoprotein(a) [apo(a)], the unique glycoprotein component of Lp(a), consists of tandem repeats of a plasminogen kringle (K) IV-like domain, followed by sequences homologous to the plasminogen KV and protease domains. Apo(a)/Lp(a) has been consistently shown to regulate endothelial function and inhibit plasminogen activation. In the present study, we have demonstrated that apo(a), signaling via integrin alphaVbeta3, is the functional unit in Lp(a) to stimulate in vitro endothelial cell (EC) proliferation and migration, and activate focal adhesion kinase (FAK) and mitogen-activated protein kinases (MAPK) in cultured ECs. Both apo(a) and Lp(a) have also been shown to reduce the levels of active and total transforming growth factor (TGF)-beta in cultured EC medium in an integrin alphaVbeta3–dependent manner. Despite the stimulatory effects of apo(a) on EC proliferation and migration, we have further confirmed an inhibitory effect of apo(a) on EC in vitro angiogenesis using a fibrin gel tube formation assay. We have provided evidence proving apo(a) inhibits angiogenesis through inhibition of plasminogen activation, and this inhibitory effect is dependent on the presence of apo(a) KV domain. Lastly, apo(a) is shown to reduce the protein levels of annexin A2 and S100A10 in ECs, which implies another potential mechanism by which apo(a)/Lp(a) could impair plasminogen activation on cell surface. In summary, we have discovered the first complete outside-in signaling pathway elicited by apo(a)/Lp(a) in ECs and have built up a connection between the ability of apo(a) to inhibit plasminogen activation and its inhibition of angiogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-25 18:29:47.106

No description available.

The regulated interaction of leukocyte with endothelium is of key importance during normal immune surveillance and leukocyte infiltration to sites of infection in the inflammatory response. This thesis is concerned with the structure and function of CD31 (platelet-endothelial cell adhesion molecule-1), one of the adhesion molecules implicated in these processes. Previous work has shown both in vivo and in vitro that CD31 is involved in the final step of leukocyte recruitment, transmigration across the endothelial monolayer. CD31 mediated adhesion is complex, since it is capable of mediating multiple adhesive interactions, both to itself (homophilic adhesion) and to other ligands (heterophilic adhesion). In order to study homophilic adhesion, an heterologous cell-protein assay was used in combination with recombinant chimeric CD31Fc fusion proteins, ICAM-3/CD31 chimeras and chimeras between human and murine CD31. These reagents located the homophilic binding site to the NH₂-terminal domains 1 and 2, but also define a non-binding accessory role for the membrane proximal domains. Using site-directed mutagenesis to target all of the exposed charged residues in domain 1 and a subset of charged residues in domain 2, five residues were identified, mutations in which resulted in inhibition of homophilic adhesion. These residues map to both faces of the domain 1 immunoglobulinlike fold, suggesting that each molecule of CD31 interacts with two others. A novel zipper model of homophilic adhesion involving CD31 lateral association analogous to that seen amongst cadherins is proposed on the basis of these results. Evidence for lateral association of CD31 to form dimers was obtained from biophysical, biochemical and molecular biology techniques. These show that Cd31 exists in an equilibrium between monomeric and dimeric forms both in solution as soluble recombinant protein, and at the cell surface. In solution the affinity of the interaction was calculated to lie in the range 12-14μM. A large panel of anti-CD31 monoclonal antibodies were generated and tested for their ability to effect homophilic adhesion. Inhibitory antibodies were identified, mapping throughout the extracellular domain, away from the ligand binding site. In addition possible stimulating antibodies mapping to the membrane proximal domains were also identified. This indicates that CDS 1 may be induced to undergo conformational changes which effect homophilic adhesion, and it is proposed that these conformational changes may be linked to the ability of CD31 to form laterally associated dimers. Using the reagents described above, a screen of haematopoietic cell lines identified a novel heterophilic interaction, which was shown to be mediated by the integrin αvβ3. Proteinprotein assays were used to confirm a direct physical association between CD31 and αvβ3, and to map the integrin binding site to the third immunoglobulin-like fold of CD31. The functional significance of this interaction was assessed in neutrophil transmigration assays, in which both anti-CD31 and anti-αvβ3 antibodies were found to partially inhibit neutrophil transmigration.

Methylglyoxal (MG) is a reactive dicarbonyl derived as a by-product of glycolysis. If MG is not metabolized by glyoxalase-1 (Glo1), it glycates macromolecules producing advanced glycation end products (AGEs); these have been linked to larger infarct sizes and poorer cardiac function after myocardial infarction (MI). Proteins of the extracellular matrix (ECM) are prime targets for glycation by MG, but it is unknown if MG modification of the ECM may be a mechanism that contributes to the poor repair and function of the post-MI heart. This study sought to examine if MG-induced modifications of ECM proteins negatively affect fibroblast and endothelial cell function. Analysis with an MG-derived hydroimidazolone 1 (MG-H1) antibody confirmed MG modification of laminin and collagen type (Col) 1, 3, and 4. MG modifications decreased endothelial cell (EC) adhesion on Col3, Col4, and laminin and angiogenesis on ECMatrix. Furthermore, alpha smooth muscle actin staining indicated increased myofibroblast differentiation of fibroblasts on MG-modified proteins. Following induction of MI, extracted mouse hearts were decellularized and compared to healthy controls. Perhaps a result of technical challenges, both western blot and immunohistochemistry contrasted previous data by displaying a marked decrease in MG-H1 modifications post-MI. Overall, these results indicate that MG modifications of the ECM negatively influence EC and fibroblast function, requiring more research on their impact in cardiovascular disease progression.

No description available.

Diabetic retinopathy (DR) is an incurable, chronic disease that is the leading cause of blindness in working-age adults. A prominent characteristic of DR is the extensive dysfunction within the retina microvasculature. Specialized vascular cells known as pericytes (PCs) are lost or become dysfunctional during disease progression; a thickening of the extracellular matrix (ECM) composing the vascular basement membrane (vBM) and endothelial cell (EC) tight junction disruption are also key features of this disease and contribute to its pathogenesis. PC loss is believed to be a central cue for disease initiation. However, studies inducing PC loss and observing acute changes in the vasculature did not report severe vessel damage or vBM thickening, suggesting that the effects of PC loss occur over a longer period of time. Because the chronic effects of PC loss are more difficult to ascertain, especially in a complex condition such as DR, the mechanisms underlying microvascular defects in DR remain poorly understood. The work presented in this dissertation focuses on pericyte-endothelial cell interactions and their interplay with the ECM/vBM during a variety of physiological and pathological conditions. First, we isolated and functionally validated a primary mouse embryonic PC cell line that we then applied to a co-culture model with ECs to better understand the dynamic interactions between these two critical components of the capillary wall. In the co-culture model, we found that primary PCs promoted EC organization into vessel-like structures and enhanced EC-EC junctions. To complement these in vitro studies, we analyzed animal models and human tissue for the PC-EC interactions and ECM/vBM remodeling under different conditions (physiological and pathological). Moreover, we analyzed microglia and astrocytes to enhance our understanding of the tissue-vessel interface, bolstering our experimental results and facilitating the generation of more hypotheses for future research. Overall, our work suggests that PC-EC interactions in diabetic scenarios play a crucial role in ECM/vBM remodeling; engagement with the ECM/vBM in turn impacted PC behaviors including migration away from the endothelium and induced EC loss of tight junctions, key changes in the onset and progression of DR. / Doctor of Philosophy / Diabetic retinopathy is a group of eye diseases occurring in patients suffering from diabetes and is the leading cause of adult blindness among the working-aged. About one in three people with diabetes over the age of 40 have overt signs of DR. The primary cause for this disease is long-term, high blood sugar levels that damages blood vessels systemically as well as in the eye. Current treatments for DR can prevent the condition from getting worse, but no treatment exists that results in a complete cure. This work described in this dissertation focuses on the interactions between vascular pericytes and endothelial cells, two of the main cell types that compose capillaries (i.e. the smallest blood vessels important for oxygen delivery). The studies presented herein also focus on the response of these cells to the extracellular matrix, a scaffold of proteins that surround pericytes and endothelial cells to stabilize blood vessels. We found that extracellular matrix components dramatically increase as a result of the interactions between pericytes and endothelial cells exposed to diabetic conditions. These changes in the extracellular matrix also had important effects on pericytes and endothelial cells and their engagement with their environment and other cells. Taken together, our work suggests that pericyte-endothelial cell interactions and their crosstalk with the ECM play an important role in blood vessel formation and in the accumulation of microvascular defects that fuel diabetic retinopathy progression.

Non-hematopoietic stromal cells (SCs) promote and regulate adaptive immunity through numerous direct and indirect mechanisms. SCs construct and support the secondary lymphoid organs (SLOs) in which lymphocytes crawl on stromal networks and inspect antigen-presenting cells for surface-display of cognate antigens. SCs also secrete survival factors and chemotactic cues that recruit, organize, and facilitate interactions among these leukocytes. They influence antigen access by secreting and ensheathing extracellular matrix-based conduit networks that rapidly convey small, soluble lymph-borne molecules to the SLO core. Furthermore, lymph node stromal cells (LNSCs) directly induce \(CD8^+\) T cell tolerance to peripheral tissue restricted antigens and constrain the proliferation of newly activated T cells in these sites. Thus, stromal-hematopoietic interactions are crucial for the normal functioning of the immune system. LNSCs are extremely rare and difficult to isolate, hampering the thorough study of their biology. In order to better understand these stromal subsets, we sorted fibroblastic reticular cells (FRCs), lymphatic endothelial cells, blood endothelial cells, and podoplanin \(^−CD31^−\) cells (double negative stromal cells; DNCs) to high purity from resting and inflamed murine lymph nodes. We meticulously analyzed the transcriptional profiles of these freshly isolated LNSCs as part of the Immunological Genome Project Consortium. Analysis of the transcriptional profiles of these LNSC subsets indicated that SCs express key immune mediators and growth factors, and provided important insights into the lymph node conduit network, FRC-specialization, and the DNC identity. Examination of hematopoietic and stromal transcription of ligands and cognate receptors suggested complex crosstalk among these populations. Interestingly, FRCs dominated cytokine and chemokine transcription among LNSCs, and were also enriched for higher expression of these genes when compared with skin and thymic fibroblasts, consistent with FRC-specialization. LNSCs that were isolated from inflamed lymph nodes robustly upregulated expression of genes encoding cytokines, chemokines, antigen-processing and presentation machinery, and acute-phase response molecules. Little-explored DNCs showed many transcriptional similarities to FRCs, but importantly did not transcribe interleukin-7. We identified DNCs as consisting largely of myofibroblastic pericytes that express integrin \(\alpha 7\). Together these data comprehensively describe the transcriptional characteristics of four major LNSC subsets isolated from resting and inflamed SLOs, offering many avenues for future study.