Worldwide, people are being exposed to natural and man-made sources of radiation. Epidemiological studies have shown an increased risk of vascular diseases in populations that have been exposed to ionizing radiation. Vascular endothelium is implicated as one of the targets for radiation leading to the development of cardiovascular diseases. However, the molecular mechanisms behind the development of radiation-induced cardiovascular disease in acute or chronic exposed people are not fully elucidated. The hypothesis that chronic low dose rate ionizing radiation accelerates the onset of senescence of primary human umbilical vein endothelial cells has been tested in papers I and II presented in this thesis. In vitro studies show that, when exposed to continuous low dose rate gamma radiation these cells enter premature senescence much earlier than non-irradiated control cells. Quantitative proteomic analysis using isotope coded protein labeling coupled to LC-ESI-mass spectrometry and followed by protein network analysis identified changes in senescence-related biological pathways including cytoskeletal organisation, cell-cell communication and adhesion, and inflammation influenced by radiation. Moreover, the role of PI3K/Akt/mTOR pathway was implicated during the senescence process. Thus, chronic low dose rated endothelial senescence may contribute to increased risk of radiation-induced cardiovascular disease. Paper III analyse the long-term effects of local high doses of radiation to the heart using a mouse model. The results from proteomic and bioinformatics analysis indicated that an impaired activity of the peroxisome proliferator-activated receptor-alpha (PPARA) is involved in mediating the radiation response. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPARA. This was reflected by the decreased expression of PPARA target genes involved in energy metabolism and mitochondrial respiratory chain. This in vivo study suggests that alteration of cardiac metabolism contributes to the impairment of heart structure and function after radiation. Taken together, these in vitro and in vivo studies provide novel information on the pathways in heart and endothelial cells that are affected over longer periods of time by ionizing radiation.

Tumors are composed not only of malignant cells, but also of various types of normal cells, including vascular cells and infiltrating immune cells, which drive tumor development and progression. The tumor vasculature is abnormal and dysfunctional due to sustained tumor angiogenesis driven by high levels of pro-angiogenic factors. Proteins differentially expressed in tumor vessels affect vascular function and the tumor microenvironment and may serve as targets for therapy. The tumor is also a site of sustained chronic inflammation. The recruitment and activation of inflammatory cells significantly influence tumor progression and regression. Targeting molecules regulating tumor angiogenesis and inflammation in the tumor microenvironment is therefore a promising strategy for the treatment of cancer. This thesis is aiming to understand and investigate the molecular regulation of these two processes in tumors. αB-crystallin is a heat shock protein previously proposed as a target for cancer therapy due to its role in increasing survival of tumor cells and enhancing tumor angiogenesis. In this thesis, we demonstrate a novel role of αB-crystallin in limiting expansion of CD11b+Gr1+ immature myeloid cells in pathological conditions, including tumor development. In addition, we show that αB-crystallin regulates leukocyte recruitment by promoting expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin during TNF-α-induced endothelial activation. Therefore, targeting of αB-crystallin may influence tumor inflammation by regulating immature myeloid cell expansion and leukocyte recruitment. Abnormal, dysfunctional vessels are characteristic of glioblastomas, which are aggressive malignant brain tumors. We have identified the orphan G-protein coupled receptor ELTD1 as highly expressed in glioblastoma vessel and investigated its role in tumor angiogenesis. Interestingly, deficiency of ELTD1 was associated with increased growth of orthotopic GL261 glioma and T241 fibrosarcoma, but did not affect vessel density in any model. Further investigation is warranted to evaluate whether ELTD1 serves a suitable vascular target for glioblastoma treatment. Anti-angiogenic drugs targeting VEGF signaling is widely used in the clinic for various types of cancer. However, the influences of anti-angiogenic treatment on tumor inflammation have not been thoroughly investigated. We demonstrate that VEGF inhibits TNF-α-induced endothelial activation by repressing NF-κB activation and expression of chemokines involved in T-cell recruitment. Sunitinib, a small molecule kinase inhibitor targeting VEGF/VEGFR2 signaling increased expression of chemokines CXCL10, CXCL11, and enhanced T-lymphocyte infiltration into tumors. Our study suggests that anti-angiogenic therapy may improve immunotherapy by enhancing endothelial activation and facilitating immune cell infiltration into tumors.

Previous work in our laboratory indicates that the diameter of brain arteries and arterioles can be increased by N-methyl-D-aspartate (NMDA) receptor activation. We looked for expression of NMDA receptors and endothelial cell responses to NMDA receptor agonists and antagonists in the mouse brain endothelial cell line bEnd.3. Using RT-PCR and Western blotting we found evidence supporting the presence of NMDA receptor subunits NR1 and NR2C. Treatment of bEnd.3 cells with combinations of 100 μM glutamate and D-serine significantly increased intracellular calcium. However, we saw no direct evidence that NO was produced in response to NMDA receptor activation using the Griess method. We did observe an NMDA receptor-dependent increase in protein nitrosylation. This increase is unlikely related to enhanced NO levels since it was not correlated with NO production and was not inhibited by the endothelial NO synthase inhibitor L-NIO.

Angiogenesis has a crucial influence on tumour development and identification of microvessels in malignant breast cancer tissue is an indicator for worse prognosis. Angiogenesis is partially governed by the family of vascular endothelial growth factors (VEGF) and their receptors, by which the VEGF-A-protein seems to be the most important factor.     The aims of this work were to first establish a method for immunohistological (IH)-staining of the VEGF-A-protein for clinical use and then to label and evaluate the expression of this protein in 31 Nottingham Histology Graded (NHG I-III) breast carcinoma.      Formaldehyde-fixated tissues from invasive breast neoplasms and control tissues were labelled with monoclonal antibodies against VEGF-A and CD31-proteins using a semi-automated IH-system from Ventana BenchMark. Positively stained vessels were counted from digital copies of microscopic pictures related to mm2 tissue.     A method of IH-labelling with VEGF-A protein was successfully established before staining of the breast tissue and in 19 of the 31 breast cancers. Vessels were counted for both antibodies. The VEGF-A-antibody stained 2.7 ± 2.3 (mean ± SD) vessels/mm2 and the CD31-antibody stained 27.3 ± 19.3 in the breast carcinoma tissue. The percent of VEGF-A-stained vessels in relation to CD31-stained were 7.6% in the NHG-I- (n=3), 7.8% in the NHG-II- (n=10) and 15.0% in the NHG-III-group (n=6).     The results demonstrate that increased NHG-grade and lower differentiation can be associated with higher percent of vessels expressing VEGF-A-protein. The result was not statistically certified because of the small number of stained breast cancers and additional investigations are recommended before clinical use.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

Endothelial cells (ECs) respond to flow but the exact mechanism producing alignment is not completely understood. We characterized EC alignment in microfluidic channels, 4 mm wide by 350 um high, to generate shear of 20 dynes / cm2 across the cell surface. In microchannels, ECs aligned perpendicular under flow. Analytical tools were developed to quantify nuclear alignment at 67% for human umbilical vein endothelial cells (HUVECs); cell elongation under shear flow shifted aspect ratio from 2.41 to 2.86. We next sought to probe the mechanism through which ECs communicate during realignment. The planar cell polarity (PCP) signaling pathway is involved in cell organization and coordination during development. A number of genes are known to affect the formation and organization of cellular structures through PCP signaling in human ECs. Higher expression of Vangl1 and Dvl1 proteins did not alter cell reorganization; knockdown of Vangl1 expression decreased EC alignment.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

Endothelial cells (ECs) respond to flow but the exact mechanism producing alignment is not completely understood. We characterized EC alignment in microfluidic channels, 4 mm wide by 350 um high, to generate shear of 20 dynes / cm2 across the cell surface. In microchannels, ECs aligned perpendicular under flow. Analytical tools were developed to quantify nuclear alignment at 67% for human umbilical vein endothelial cells (HUVECs); cell elongation under shear flow shifted aspect ratio from 2.41 to 2.86. We next sought to probe the mechanism through which ECs communicate during realignment. The planar cell polarity (PCP) signaling pathway is involved in cell organization and coordination during development. A number of genes are known to affect the formation and organization of cellular structures through PCP signaling in human ECs. Higher expression of Vangl1 and Dvl1 proteins did not alter cell reorganization; knockdown of Vangl1 expression decreased EC alignment.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.