No description available.

Otolin-1 is a collagenous, C1q domain-containing extracellular matrix protein that has been identified and characterized in the inner ear. Recently, mass spectrometry analysis of left ventricular cardiac tissue detected a peptide of Otolin-1. Experimental analysis confirms Otolin-1 is an insoluble protein present in the left ventricular extracellular matrix whose expression decreases dramatically post-myocardial infarction beginning at day 5 post-MI. The protein is localized to the gap junctions of cardiac myocytes, and depletion of protein levels in the infarcted region of the left ventricle shows strong association with ventricular dimensions, observed via echocardiography.

An alternative gene therapy method, non-autologous somatic-gene therapy, is the use of a genetically modified universal cultured cell line that can be implanted into different allogeneic recipients. When used as recombinant cells in microcapsules, myoblasts possess several advantages over other cell types, namely their ability to terminally differentiate thus preventing overcrowding within the capsular space. However, encapsulated myoblasts demonstrate decreased proliferation and myogenic differentiation when compared to unencapsulated myoblasts due to the unnatural capsule environment. This study aims to improve the microcapsule environment by incorporating basic fibroblast growth factor (bFGF) and insulin-like growth factor-11 (IGF-11) and the extracellular matrix materials, collagen, laminin-1 and merosin (laminin-2) within the microcapsules in an attempt to mimic the natural surrounding required for myoblast growth and differentiation. While bFGF lead to significant increases in encapsulated myoblast proliferation, it did not appear to be an ideal choice for optimizing the microcapsule environment due to its inhibitory effect on differentiation and the relative cost in therapeutic delivery of proteins. Both merosin and the combination of laminin and merosin together provide a better alternative for increasing myoblast growth and survival within microcapsules since they have no apparent inhibitory effect on myogenic differentiation, and produce similar proliferative results seen when using bFGF. In terms of differentiation, the addition of IGF-11 to the microcapsules or the use of a myoblast cell line overexpressing IGF-11, aid in increasing the myogenic differentiation of encapsulated myoblasts, however, differentiation levels still do not approach those seen in unencapsulated myoblasts. The positive results obtained with the growth factors and matrix materials employed in this study are important steps towards the optimization of microcapsules by improving both the proliferation and differentiation of encapsulated myoblasts. However, more study is needed to elucidate possible solutions to the continued problem of decreased differentiation of myoblasts within APA microcapsules in order to achieve myogenic differentiation that is comparable to what is seen in unencapsulated myoblasts. / Thesis / Master of Science (MS)

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.

Rapid rearing of dairy heifers during late prepuberty has been linked to impairments in mammary development and reductions in milk yield. Our objective was to determine how varying energy intakes between 2 and 14 wk of age affect mammary parenchymal development. At 2 wk of age, Holstein calves were assigned to 1 of 4 treatments (HH, HL, LH, and LL) with 2 levels of energy intake (High or Low) and 2 periods of growth (2 to 8 and 8 to 14 wk of age). At 14 wk, parenchyma at the stromal interface, mid-gland, and above the cistern were collected, fixed, and embedded in paraffin. Digital images of stained sections were used to determine tissue composition (% epithelium, lumen, and stroma). Immunochemistry revealed estrogen receptor (ER) and Ki67 (nuclear proliferation antigen) positive cells, type IV collagen, fibronectin (FN), and laminin. Images representing 4 increasing grades were used to quantify ECM protein deposition. Lumenal and stromal areas were 3.5 ± 1.4% higher (p<0.01) and 4.0 ± 1.7% lower (p<0.01), respectively, in HH and HL heifers. Ki67 labeling in terminal ductular units and subtending ducts was 2.1 ± 0.8% (p<0.01) and 1.4 ± 0.7% (p<0.05) lower for the same feeding level combination. FN deposition was also increased (p<0.05) in HH and HL heifers. High rates of gain between 2 and 14 wk of age resulted in greater lumenal area and reduced cell proliferation in mammary parenchyma at 14 wk of age. Changes in FN deposition could have mediated growth differences. / Master of Science

Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 129-157). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 129-157). Also available on the Internet.