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The corneal wound healing response involves the activation of keratocytes to proliferate from a quiescent phenotype. The mitogens that cause the initial transformation of the quiescent keratocytes to the active phenotype have not been identified. Even though serum is commonly used to replicate this in vitro, the cornea is avascular and therefore likely not exposed to serum. In the first part of this dissertation, a DMEM/F12 extract of corneal stromas was made and shown to stimulate keratocyte proliferation in both a dose-dependent and cell-density dependent manner. This extract contains mitogens that differ from the mitogens present in serum based on their effect on keratocytes and their biochemical characteristics. Culture in extract replicates in vitro the changes observed during the activation of keratocytes in the wound-healing phase.The corneal stroma contains an extensive extracellular matrix that consists primarily of collagens and proteolgycans. This matrix is maintained and secreted by the keratocytes, cells with unique characteristics lost during the activation observed at wound healing. The second part of this dissertation aims to develop a defined culture medium that maintains the keratocyte phenotype during proliferation. Keratocytes were cultured in serum-free medium and the effect of the growth factors on the markers for the keratocyte phenotype determined. Only insulin was shown to stimulate cell proliferation in a consistent manner, while maintaining commonly accepted keratocyte markers. When this defined culture medium was supplemented with ascorbic acid to study collagen synthesis, a marked increase in both collagen synthesis and keratan sulfate proteoglycan accumulation was measured. This newly developed culture medium, containing insulin and ascorbate, allows for cell growth, maintains the keratocyte markers, and could be used to study the native, non-activated keratocyte phenotype in culture.This dissertation shows that the culture media described herein replicate in vitro all the phenotypes observed during the corneal wound healing response in vivo. These culture media, in turn, could be used to obtain more knowledge about the different keratocyte phenotypes, and how they could be manipulated in culture. (329 words)

Background: The cornea is the outermost transparent layer of the eye and it is responsible for the majorityof the eye’s total focusing power. Keratocytes are the resident cells of the corneal stroma and their function isto produce extracellular matrix components and to take part in corneal healing after injury, which may occurdue to trauma, infection or surgery. The process of corneal wound healing is complex. Shortly, keratocytesadjacent to the corneal wound undergo apoptosis and remaining cells start the process of proliferation andmigration in order to close the wound. Next, an influx of inflammatory cells such as macrophages andneutrophils occurs in order to clear the cornea from cellular debris. The final stage of the healing processrestores the quiescent state of keratocytes and remodels any disordered extracellular matrix components,leading to a healthy, transparent cornea. However, when the process of corneal wound healing is incompleteor disturbed, corneal scarring may occur, which can lead to significantly impaired vision. Despite extensiveresearch on corneal wound healing, corneal scarring remains a major cause of preventable blindness. Thehealing process is dependent on various cytokines and growth factors. However, it is possible that also othersignal substances are involved. Substance P (SP) is a neuropeptide well known for its role in pain perception.It has been shown that SP can also be produced by non-neuronal cells, including cells of the cornea, and thatit can have vast effects on physiological functions, including immune cell activity, and cellular processes, suchas cell migration, proliferation, and production of proinflammatory cytokines. Similarly, acetylcholine (ACh),a classical neurotransmitter, has also been reported to be produced by non-neuronal cells, including cornealepithelium, and to be involved in cell proliferation, angiogenesis, cell migration, apoptosis, and collagen geneexpression. In the studies of this thesis, it is hypothesized that neuropeptides and neurotransmitters areproduced by human keratocytes and that this production is increased in response to corneal injury. Moreover,it is hypothesized that the non-neuronal SP and ACh produced by injured keratocytes participate in cornealwound healing by enhancing keratocyte migration and proliferation, and/or by decreasing keratocyteapoptosis. The aims of this thesis project were to test these hypotheses and to study the underlying inter- andintracellular mechanisms of the effects of SP and ACh on keratocytes.Results: Cultured primary cells of the human corneal stroma expressed keratocyte markers (keratocan,lumican, CD34, and ALDH), the tachykinins SP and NKA, catecholamines (adrenaline, noradrenaline anddopamine), ACh, and glutamate. Moreover, the cells expressed neurokinin-1 and -2 receptors (NK-1R andNK-2R), dopamine receptor D2, muscarinic ACh receptors (mAChRs) M1, M3, M4 and M5, and NDMAR1glutamate receptor. Significant differences were observed between expression profiles in cultured keratocytesobtained from central and peripheral cornea. Such differences could also be seen between keratocytescultured under various serum concentrations. Expression and secretion of SP in cultured keratocytes wasincreased in response to injury in vitro. SP enhanced migration of cultured keratocytes through stimulation ofits preferred receptor, the NK-1R, and activation of the phosphatidylinositide 3-kinase and Rac1/RhoApathway and subsequent actin cytoskeleton reorganization and formation of focal adhesion points. Moreover,SP stimulation led to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8(IL-8), which also contributed significantly to SP-enhanced keratocyte migration and to attractingneutrophils. ACh enhanced keratocyte proliferation in vitro at low concentrations and this stimulation wasmediated through activation of mAChRs and activation of MAPK signalling. Moreover, ACh stimulation led toupregulation of two proliferation markers: PCNA and Ki-67. ACh was also able to protect cultured keratocytesfrom Fas-induced apoptosis, even at low concentrations. Activation of mAChRs was necessary for this latterprocess to occur. ACh reduced caspases 3/7 activation in Fas-treated keratocytes. Inhibition of the PKB/Aktpathway revealed that its activation is essential for mediating the anti-apoptotic effect of ACh in keratocytes.Conclusions: This thesis shows that human keratocytes express an array of neuropeptides (SP, NKA) andneurotransmitters (ACh, adrenaline, noradrenaline, dopamine and glutamate), and their receptors, and thatstimulation of NK-1R by SP and stimulation of mAChRs by ACh lead to keratocyte cellular processes that areknown to be involved in corneal wound healing. Specifically, SP enhances keratocyte migration throughupregulation of IL-8, ACh enhances keratocyte proliferation through activation of the MAPK signallingpathway, and ACh is able to protect keratocytes from apoptosis by activation of the PKB/Akt pathway. Takentogether, these findings suggest that both SP and ACh, if entered at the proper stage, could be beneficial forcorneal wound healing.

Breast cancer is the most common cancer in female population worldwide. In addition to mutations, the breast tumor microenvironment especially the tumor cell - stroma interactions through extracellular matrix components and multiple growth factors have been shown to promote tumor progression. Among those, increases in both TGF-β (transforming growth factor beta) activities and periostin expression were associated with tumor cell survival, proliferation and metastasis. TGF-β role in breast cancer progression including its ability to promote periostin expression has been extensively studied. In contrast, the role of periostin in cancer progression remains to be fully understood. Thus, the present study aimed to determine whether TGF-β and periostin have effect on each other’s expressions in breast tumor and stroma cells using in vitro cell models. Through Western blot analyses and ELISAs, the periostin and TGF-β expressions of both stroma and tumor cells were analyzed following TGF-β and periostin treatments, respectively. The results indicate that TGF-β treatments led to significant increase in periostin expression in fibroblasts (p<0.05). In addition, periostin was differentially expressed by human breast cancer cells following TGF-β1 treatment. The TGF-β activities involved activation of pSMAD2 in both L929 fibroblasts and MCF10A mammary cells. Taken together, all experimental data indicate that within the breast tumor TGF-β and periostin likely participate in a regulation loop. Whether this putative regulation loop is critical to metastasis remains to be determined. Should periostin play a critical role in breast cancer progression, it could become a specific target in the preventive and/or therapeutic development of breast cancer patients.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

Cancer is a devastating condition, yet its prevalence is not surprising when one considers the possibility that growth and motility define the default state of epithelial cells. What is more surprising is that epithelial cells can be induced to a “fragile quiescent state” in multicellular organisms through constant inhibitory influences from extracellular sources. In other words, the immotile and growth-constrained behavior we associate with epithelia (quiescence) is not the default state cells in a multicellular organism, but rather must be exogenously induced by tissue-specific (and systemic) factors. Quiescence therefore, is a fragile existence for any cell, as the removal of differentiating signals should cause the cell to revert to a migratory, stem-like default state. It will be argued that cancer is better understood as a disease of tissues rather than individual cells, and the complexities of tissues cannot be inferred from the study of cells in isolation. In-vitro studies which have been used to explain carcinogenesis will be critically reviewed and their relevance to in-vivo conditions will be questioned. Complex signaling mechanisms define the relationship between the epithelium and other cells in a metazoan tissue. These signals originate both from the stromal/mesenchymal compartments and between epithelial cells. Studies have shown that carcinogenic insults which affect the stroma alone can turn an otherwise normal epithelium cancerous, while transplanting “cancerous” epithelial cells into an otherwise normal stroma does not result in neoplasm formation. Experimental evidence has confirmed that the differentiation fate of any epithelial cell is malleable depending on its environmental context. Further, it was previously thought that epithelium had to dramatically change identity in order to be capable of migration. It will be shown that collective motility is an endogenous capability of epithelial cells, and multiple non-mammalian and mammalian in-vivo studies of collective epithelial motility will be reviewed to better understand epithelial motility in cancer. In fact, it will be shown that the creation of adhered epithelial sheets requires the same morphological changes necessary for motility. Lastly, evidence that heterogeneous cell populations contribute to epithelial cell migration in development and metastasis will be presented.

Neuroblastoma, a neuroendocrine tumor, is the most common cancer in infancy. 75 % of those affected are under the age of 5. The disease is heterogeneous and survival rate is low.   Current treatment of neuroblastoma consists of surgery, radiation and chemotherapy, where the targets for the treatment are the malign cells. Due to the cancer cells instable genome there is a risk for resistance development. This negatively impacts the treatments goal of hindering tumor growth and spread.  Tumor growth is not only determined by malign cells but also the interactions of those tumor cells with tumor vessels and different types of cells in the tumor stroma.   The aim of this paper is to develop a relevant histological method to study the properties of tumor stroma in tumor sections retrieved from human NB tumor xenografts in mice treated with angiogenesis inhibitors SU11657 and Zoledronic acid. The study is a continuation of previous studies with the inhibitors which have shown good effect on tumor growth and angiogenesis on neuroblastoma.   In the short term treatment with SU11657 and Zoledron acid showed that tumor growth declined. In the longer treatment with SU11657 the growth didn’t decline with the same rate compared to the short term treatment. Angiogenesis on the other hand decreased in all the treatments independent of treatment duration. The histological staining with Sirius red revealed that treated tumors had an increased amount of stroma compared to the untreated tumors.   In conclusion the relative increase of tumor volume, decreased number of vessels and expansion of tumor stroma in the longer treatment with SU11657 indicated that tumors might survive the angiogenesis inhibitor treatment through expansion/activation of its stroma. The histological staining with Sirius red in saturated picric acid marked the collagen, i.e. stroma, well and enabled quantification of the stroma.

Ces dernières années, il est apparu que le stroma péritumoral et en particulier les fibroblastes associés au cancer exprimant l’actine musculaire lisse (CAFs : carcinoma-associated fibroblasts/ myofibroblasts) faisaient partie intégrante du processus carcinologique observé dans diverses tumeurs épithéliales y compris dans les carcinomes mammaires.L’objectif de ce travail de thèse a donc été :1) D’analyser la distribution « in situ » de ses CAFs/myofibroblastes dans les lésions mammaires de carcinome intraductal (CID), de carcinomes invasifs non spécifiques (No Special Type – CaNST) et de carcinomes métastatiques.2) De déterminer si cette réaction myofibroblastique pouvait prendre naissance à partir des fibroblastes « résidents » CD 34 positifs par l’intermédiaire du TGF-1 et de son récepteur.3) De déterminer si l’action pro carcinogénique de cette réaction myofibroblastique pouvait être liée à la surexpression stromale de la métalloprotéinase de la matrice de type-2 (MMP-2).4) De déterminer si l’expression des récepteurs aux glucocorticoïdes (GR) était présente au sein de ces cellules stromales afin de constituer une cible thérapeutique potentielle. Nos trois premiers travaux ont consisté en une analyse semi-quantitative par immunohistochimie de l’expression de l’anticorps anti- CD 34, marqueur de fibroblastes mammaires, et de l’anticorps anti–actine musculaire lisse (AML), marqueur de myofibroblastes, au sein du stroma de lésions CID, de CaNST et de lésions métastatiques, hépatiques et ganglionnaires. Ces expériences ont mis en évidence la perte progressive de l’expression stromale du CD34 et l’apparition progressive, « en miroir », de l’expression stromale de l’AML autour des foyers de CID et ce de manière proportionnelle au grade nucléaire de ces lésions. En ce qui concerne les CaNST, nous avons constaté la perte de l’expression stromale du CD34 et la présence de la réaction myofibroblastique AML positive dans 100 % des cas. L'intensité d'expression de l'actine musculaire lisse était corrélée de manière significative avec la présence de métastases ganglionnaires. Enfin, cette réaction myofibroblastique a été également retrouvée de manière constante au sein des métastases ganglionnaires et hépatiques. Notre quatrième travail a consisté en l’étude immunohistochimique du TGF-1 et de son récepteur (TGF-R1) dans les lésions de CaNST et au traitement in vitro de lignées cellulaires de fibrocytes mammaires par du TGF-1. Nous avons pu constater une augmentation statistiquement significative de l’expression du TGF-1 au sein des cellules tumorales et une augmentation statistiquement significative de l’expression du TGF-R1 au sein du stroma péritumoral par rapport au tissu mammaire normal. Par ailleurs, le traitement in vitro d’une lignée de fibroblastes mammaires par le TGF-1 faisait apparaître une expression d’AML au sein de ces cellules.Notre cinquième travail a permis de mettre en évidence, par immunohistochimie, une relation entre l’expression stromale de la métalloprotéinase de la matrice de type 2 (MMP-2) et le sous-type tumoral selon la classification luminale. Les tumeurs exprimant HER-2/Neu présentaient une plus forte expression stromale de MMP-2, considérée comme potentiellement pro-invasive.Enfin, notre dernier travail a mis en évidence l’expression fréquente (>90% des cas) des récepteurs aux glucocorticoïdes (GR) au sein des CAFs/ myofibroblastes des CaNST. Cette expression était corrélée de manière significative avec le grade tumoral, l’indice de Ki-67 et l’expression de ces mêmes GR au sein des cellules tumorales épithéliales.En conclusion, les résultats découlant de ces différents travaux démontrent une réaction stromale péritumorale constante et ce tout au long du processus de développement tumoral, dès le stade de carcinome in situ jusqu’au stade métastatique.Une des origines potentielles de ces myofibroblastes est vraisemblablement la transformation des fibroblastes « résidents » mammaires par l’intermédiaire du TGF- et de son récepteur. Ces myofibroblastes présentent par ailleurs des caractéristiques d’expression de MMP-2 différentes en fonction des sous types de carcinomes mammaires suggérant que le stroma péritumoral est en partie différent en fonction du sous type tumoral. Enfin, les récepteurs aux glucocorticoïdes (GR) qui sont fréquemment observés dans ces cellules myofibroblastiques pourraient à l’avenir constituer de nouvelles cibles dans la régulation de l’action de ces cellules. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished