Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular cells, such as endothelial cells (ECs). Hereditary (H) PAH is mainly caused by ―loss-of-function‖ mutations in the gene coding for the bone morphogenetic protein type II receptor (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. The hypothesis of this thesis was that BMPR2 mutations produce an imbalance in EC protein expression and/or activity that is integrally related to the development of abnormalities in lung vascular function and structure in HPAH. Patient-specific blood-outgrowth endothelial cells (BOECs) expanded ex vivo from peripheral blood mononuclear cells from patients with HPAH and healthy subjects were used to examine the consequences of BMPR2 mutations on the BOEC protein expression profile as well as on their functionality. Functional analyses of the BOECs revealed that HPAH-derived BOECs are more susceptible to apoptosis and more proliferative compared with healthy controls. Protein isolates of BOECs from patients with HPAH and from healthy subjects were subjected to 2-D gel electrophoresis and stained for total proteins and phosphoproteins, and to a quantitative computerassisted analysis. Differentially regulated proteins were identified by mass spectrometry (LC-MS/MS). Of the 416 total proteins detected under basal conditions, 11 were significantly downregulated in HPAH-derived BOECs and 11, including the translationally controlled tumor protein (TCTP), were upregulated. TCTP has previously been shown to be involved in systemic arteriolar remodeling, inflammation and growth. Therefore, the potential role of TCTP in PAH was studied in vivo in the SU5416 rat model of severe angioproliferative PAH. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. Similarly, abundant TCTP immunostaining was also seen in human PAH lung sections, again associated with complex vascular lesions. In BOECs, TCTP was found to participate in cell growth and survival. These data suggest that TCTP could play an important role in PAH by mediating pro-survival and growth signaling in vascular cells, contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

No description available.

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.

Replicative exhaustion of endothelial cells (ECs) contributes to the pathogenesis of age-related vascular disorders, including atherosclerosis and impaired wound healing. Conversely, abnormal proliferation of ECs underlies the development of EC-derived malignancies, such as haemangioblastoma and angiosarcoma. The central objective of this thesis was to delineate mechanisms that regulate the replicative lifespan of human ECs and molecular alterations that occur during immortalisation of ECs. The gradual shortening of telomeres (chromosome-end structures) is one mechanism that restricts the replicative lifespan of human ECs. Telomere shortening initiates an irreversible growth arrest or senescence through activation of a TP53-mediated DNA damage response. Expression of the cyclin-dependent kinase inhibitor, p16INK4a, is also increased and reinforces senescence via the retinoblastoma pathway. Overexpression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity and extends the lifespan of human ECs, but is not sufficient for immortalisation. The current study demonstrated that p16INK4a repression by promoter methylation was a frequent event during immortalisation of hTERT-transduced bone marrow ECs (BMECs), occurring in 5 of 12 clones. Repression of p16INK4a concurred with the development of recurring chromosomal aberrations, which appeared to be a consequence of telomere dysfunction and chromosome fusions. Loss of p16INK4a and the development of a complex karyotype were associated with a more transformed phenotype in hTERT-immortalised BMECs. The investigations described in this thesis were the first to associate loss of p16INK4a expression with the accumulation of chromosome aberrations. Repression of p16INK4a in only a subset of immortal BMECs provided impetus for investigating whether there was a functionally analogous defect in the hTERT-immortalised BMECs that retained p16INK4a expression. In normal human cells, oncogenic Ras upregulates p16INK4a and induces senescence independently of telomere shortening. This thesis demonstrates that the immortal BMECs that retained p16INK4a expression had a defective response to oncogenic Ras, which may have contributed to the immortalisation of these cells. Whole genome and proteome analyses identified additional alterations in gene copy number and protein expression specific to p16INK4a-positive or -negative immortal BMECs. Overall, these investigations provide new insight to the potential consequences of p16INK4a repression during carcinogenesis and describe novel molecular alterations that occur during immortalisation of human ECs.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Ulm, Univ., Diss., 2008.

Universiẗat, Diss--Gießen, 2001.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Thesis advisor: Dr. William Durante. "December 2008" Includes bibliographical references.

Ulm, Univ., Diss., 2001.