In vitro cell culture is an essential part of many cell and tissue engineering approaches. In particular, monolayer culture of mammalian cells is a key tool for applications such as cell therapy. Novel bioreactors like the Cellerator(TM) allow for expansion of cell populations on mechanically stimulated surfaces coated with proteins. This thesis constitutes a preliminary study which focused on cell-matrix interactions in the absence of stretch. The aim was to establish standard protocols for protein coating on poly (dimethyl siloxane) (PDMS) and for measuring cell proliferation. Specifically, the proliferation of rat pulmonary artery vascular smooth muscle (PAC1) cells on type I collagen and soluble fibronectin was studied. Growth curves were obtained and the doubling time for subconfluent cultures was computed. Although cell-matrix interactions do not enhance proliferation of PAC1 cells, it was found that a preliminary sulphuric acid treatment is necessary to yield a well-behaved culture.

The extracellular matrix is a vital component of the cardiovascular system, in that, it not only provides structural support but also plays a critical role in the maintenance of cellular stability. One of the major components of the vascular matrix is elastin, which confers vessels with the specialized property of stretch and recoil. Elastin deficiency has been implicated in many vascular diseases and determined experimentally to be a negative regulator of smooth muscle cell proliferation. In zebrafish, two elastin genes have been identified, which are actively expressed during development. Based on this finding, protein production and spatial localization for the two elastin proteins was studied by immunohistochemistry with specific antibodies. Results revealed a global distribution for elastin 1 in the ventral aorta and swim bladder, whereas elastin 2 was preferentially localized to the bulbus arteriosus indicating a possible specialized function of elastin 2 in this structure. This observation, and the unique physiological property of this structure, suggests a possible reason for the preservation of both elastin genes during evolution. / In the second part of this study, elastin-null mice were studied to uncover the impact of the loss of elastin on the expression of other elastic fiber-associated proteins. The expression of fibrillin-1, the major component of microfibrils, was not altered in the absence of elastin, implying that elastin is not necessary for the formation of microfibrils. On the other hand, both fibulin-2 and -5 were upregulated in the absence of elastin, suggesting that expression of these genes are controlled by elastin. Overall, this study highlights the importance of elastin in evolution, as well as its potential role in the regulation of expression of other matrix molecules.

No description available.

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 2002. / Includes bibliographical references (leaves 292-352).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2010. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 101-119).

Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.

No description available.