Basic research into the histology, endocrine control, and local regulation of prepuberal ruminant mammogenesis was conducted to provide a better understanding of this important developmental period. Histologic features of prepuberal ruminant mammary parenchymal morphogenesis were examined in tissue samples taken from ewe lambs at 2 (n = 5), 3 (n = 15), 6 (n = 26), 9 (n = 7), 12 (n = 5), and 13 wk (n = 20), and from Holstein heifers at 4 (n = 1) and 6 mo (n = 2). Examination of approximately 8000 histologic sections revealed that mammary parenchymal morphogenesis in sheep and cattle occurs through the proliferation of highly arborescent ductal structures embedded in a dense stroma. These observations contrast strongly with models of mammogenesis based on murine mammary development. The formation of luminal spaces and the expansion of ducts also differed from murine mammogenesis models. Luminal spaces were shown to develop through a progressive separation of opposing sides in initially solid ductal structures. Likewise, our investigation of prepubertal ovine mammogenesis revealed that parenchymal weight, 3H-thymidine labeling, stromal weight, and parenchymal DNA were all unaffected by ovariectomy (P > 0.05), in marked contrast to the dramatic reduction in mammary development following ovariectomy in rats, mice, and heifers. Responsiveness to exogenous estrogen (0.1 mg/kg) was demonstrated by increased 3H-thymidine labeling (P < 0.05) in both intact and ovariectomized lambs. Three dimensional collagen gel cultures of bovine mammary organoids from the peripheral (OUTER) and medial (INNER) parenchymal zones were used to characterize the proliferative and morphogenetic responses to local-acting growth factors. The proliferation of OUTER cells was 2 to 3 times greater than INNER cells (P < 0.0001) in response to IGF-I stimulation. Dramatic differences in the morphology of INNER and OUTER organoids were also observed. INNER cells grew into smooth-edged colonies when treated with heifer serum but stellate colonies when treated with other mitogens. OUTER cells grew into stellate colonies regardless of mitogen treatment. These investigations highlight the fact that a great deal more research into the basic physiology of prepuberal ruminant mammogenesis is required and that dogma developed in murine model systems may not be applicable to ruminant mammary physiology. / Ph. D.

No description available.

Tendon injuries significantly impact quality of life and are often career ending. Mesenchymal stem cell (MSC) therapy is known to augment intrinsic tendon healing, however, little is known of the stem cells endogenous to tendon, the microenvironmental cues that induce tendon differentiation, and whether individual cells in an inflammatory milieu respond differently to these cues. To address these questions, a three-dimensional tenogenesis assay was developed as an efficient and reproducible metric of cellular capacity to differentiate toward tendon. In contrast to more complex assays of tenogenesis, this design incorporates a simple apparatus using commercially available plasticware for the application of uniaxial static strain in in a type I collagen cell-seeded hydrogel construct. Tendon-related gene expression, glycosaminoglycan levels, elongated cell morphologies and parallel cell alignments are enhanced with BMP-12 induction over ten days of culture. This dissertation provides novel insight to the roles of growth factors in MSC tenogenesis. Tendon healing in vivo is dependent on endogenous tendon stem cells (TSC) that mediate the inflammatory response to injury and promote synthesis of collagen and matrix remodeling, among other extracellular processes. Recent evidence suggests that these cells exist on a spectrum of differentiation potencies, and may be differently committed to the tendon fate. Individual stem cells were isolated from the tendon, and their capacities for proliferation, tri-lineage differentiation and tenogenesis were evaluated. Three distinct TSC phenotypes were revealed, and significant, positive correlations were found in quadra-differentiation potency (toward four lineages) and the expression of a strong, composite tendon phenotype. These studies have important implications in the current standard-of-care in regenerative therapies for tendon. Our benchtop tenogenesis assay can be used to determine the therapeutic potential of allogeneic MSC lines and MSCs from novel sources for 'off-the-shelf' treatments. Our study of TSCs lends valuable insight to the diversity of cell phenotypes found in tendon, and the potential contributions of each phenotype to tendon healing and homeostasis. These results further strengthen the status of tendon as a superior source of stem cells for tendon repair. / Ph. D. / Tendons are fibrous, elastic bands of collagen that connect muscles to bones and are essential to movement and proper functioning of the skeletal system. Weight-bearing tendons like the Achilles in humans and superficial digital flexor tendons in horses are particularly prone to damage and degeneration with overuse and/or aging. Bone marrow-derived stem cell treatments have shown promise in the reduction of pain and inflammation, and restoration of native tendon structure and function in cases of severe tendon injuries. However, the roles of stem cells in tendon healing, particularly their ability to transition to cell types native to tendon and integrate with an environment distinct from their own is unknown. Culturing of stem cells in three dimensional (3D) environments has enabled us to identify and understand the biochemical and mechanical signals that trigger stem cell transitions to tendon cells in tendons, but currently available 3D culture systems are complex and inefficient. In this dissertation we have developed a cost-effective and high throughput 3D culture system to assay the potential of stem cells to form tendon cells and composite tendon-like tissues. Toward this, we have also optimized the effects of known tendon proteins on the tendon fate in 3D culture of stem cells. Like most adult tissues, the tendon encompasses an in-house repository of stem cells. Tendon stem cells (TSCs) are primarily responsible for the inflammatory and reparative responses to tendon injury. Recent evidence suggests that TSCs are diverse in character, and differ from each other in their ability to form cells and tissues of fat, bone and cartilage. In this work, we provide evidence that TSCs are also differently committed to forming tendon tissue, and moreover that significant inter-relationships among gene expression patterns in these cells directly contribute to cultural diversity. In sum, our results provide novel insight to the roles of stem cells in tendon healing, particularly their response to subtle changes in their biochemical environment, and the contributions of individual cells in a milieu to a holistic reparative response.

This dissertation centers on the study of epithelial to mesenchymal cell transition (EMT) in the heart model of valve development. EMT is a process used by specific cells to invade adjacent matrix in order to differentiate into a three-dimensional structure. The first section of the project includes a study on the effects of inorganic arsenic on EMT and therefore the environmental concerns produced by deleterious effects on EMT. The second section focuses on the discovery of an intrinsic regulator of EMT, olfactomedin-1 (OLFM1). The discovery of a novel regulator of EMT in the atrioventricular canal is interesting, by itself, as it allows us to better understand the intrinsic molecular regulation of EMT in valve formation of the heart. The activity of this protein, as a regulator of cell invasion, identifies an important checkpoint in EMT. Because OFLM1 is conserved across many species, including humans, it may be a common or shared regulator of all types of EMT including cancer. Therefore, OLFM1 represents a promising new target for an anti-cancer agent as well as a potential clinical inducer of EMT to repair congenital heart disease that include valve defects.

<p>This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation.</p><p>The interstitial fluid pressure (P_IF) is of importance for control of tissue fluid balance. A lowering of P_IF <i>in vivo</i> will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling P_IF.</p><p>Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of P_IF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of P_IF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored P_IF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered P_IF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered P_IF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from <i>Streptococcus equi</i> modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction <i>in vitro</i> and control of P_IF <i>in vivo</i>, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence P_IF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.</p>

This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation. The interstitial fluid pressure (PIF) is of importance for control of tissue fluid balance. A lowering of PIF in vivo will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling PIF. Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of PIF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of PIF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored PIF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered PIF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered PIF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from Streptococcus equi modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction in vitro and control of PIF in vivo, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence PIF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses. / May 2006

No description available.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.