Mounting evidence suggests that the normal biomechanical state of arteries may include a nearly equibiaxial intramural stress, and that arteries tend to undergo rapid and dramatic remodeling when perturbed from this normal state. Technical developments in the early 1980s and late 1990s enabled in vitro and ex vivo studies, respectively, of isolated perfused microvessels, and it is clear that they share many similarities in behavior with arteries. To date, however, there has been no systematic study of the effects of biaxial loading on the biomechanical behavior of arterioles. In this project, we describe a modification to a prior in vitro arteriole test system that allowed us to investigate the role of altered axial stretch on the passive, myogenic, and fully contracted biaxial behavior of isolated rat cremaster arterioles. We show that axial stretches from 85% to 110% of normal values induce modest changes in the measured circumferential and axial stress-stretch behavior and similarly in traditional measures of distensibility and myogenic index. Nevertheless, altered axial stretch has a dramatic affect on the biaxial state of stress and it appears that near equibiaxial stress occur at axial stretches larger than those used previously. Whereas this finding will not affect prior estimates of material and functional behavior, it may have important implications for the design of long-term ex vivo and in vivo studies wherein vessel growth and remodeling are critical.

Epithelial mesenchymal/-myofibroblast transition (EMT/EMyT) has emerged as one of the central mechanisms in wound healing and tissue fibrosis. The main feature of EMyT is the activation of a myogenic program, leading to the induction of the α-smooth-muscle actin (SMA) gene in the transitioning epithelium. Recent research suggests that intercellular contacts are not merely passive targets, but are active contributors to EMT/EMyT. Indeed, our group showed previously that contact uncoupling or injury is necessary for TGFβ to induce EMyT (two-hit paradigm). Further, our previous work also revealed that Smad3, the main TGFβ-regulated transcription factor, binds to the Myocardin Related Transcription Factor (MRTF), the prime driver of SMA promoter, and inhibits MRTF’s transcriptional activity. During EMyT, Smad3 eventually degrades, which liberates the MRTF-driven myogenic program. However the mechanisms whereby cell contacts regulate the fate of Smad3 and MRTF during EMyT are poorly understood. Accordingly, the central aim of my studies was to explore the role of intercellular contacts, in particular that of Adherens Junction (AJs) in the induction of the myogenic reprogramming of the injured epithelium. This thesis describes two novel molecular mechanisms through which AJs impact EMyT. In the first part, we show β-catenin, an AJs component and transcriptional co-activator counteracts the inhibitory action of Smad3 on MRTF. Moreover we reveal that β-catenin is necessary to maintain MRTF stability via protecting MRTF from proteasomal degradation. Thus, β-catenin is an indispensable permissive factor for SMA expression. In the second part, we demonstrate that contact injury and TGFβ suppress the expression of the phosphatase PTEN. EMyT-related reduction or absence of PTEN potentiates Smad3 degradation. EMyT is associated with enhanced phosphorylation of the T179 residue in Smad3 linker region, and this event is necessary for Smad3 degradation. PTEN silencing increases the stimulatory effect of contact uncoupling and TGFβ on SMA promoter activity and SMA protein expression. Thus, the integrity of intercellular contacts regulates the level of PTEN, which in turn controls Smad3 stability through impacting on T179 phosphorylation. This new knowledge holds promises for targeted therapies and more effective prevention of the currently incurable fibroproliferative and fibrocontractile diseases.

Mounting evidence suggests that the normal biomechanical state of arteries may include a nearly equibiaxial intramural stress, and that arteries tend to undergo rapid and dramatic remodeling when perturbed from this normal state. Technical developments in the early 1980s and late 1990s enabled in vitro and ex vivo studies, respectively, of isolated perfused microvessels, and it is clear that they share many similarities in behavior with arteries. To date, however, there has been no systematic study of the effects of biaxial loading on the biomechanical behavior of arterioles. In this project, we describe a modification to a prior in vitro arteriole test system that allowed us to investigate the role of altered axial stretch on the passive, myogenic, and fully contracted biaxial behavior of isolated rat cremaster arterioles. We show that axial stretches from 85% to 110% of normal values induce modest changes in the measured circumferential and axial stress-stretch behavior and similarly in traditional measures of distensibility and myogenic index. Nevertheless, altered axial stretch has a dramatic affect on the biaxial state of stress and it appears that near equibiaxial stress occur at axial stretches larger than those used previously. Whereas this finding will not affect prior estimates of material and functional behavior, it may have important implications for the design of long-term ex vivo and in vivo studies wherein vessel growth and remodeling are critical.

Epithelial mesenchymal/-myofibroblast transition (EMT/EMyT) has emerged as one of the central mechanisms in wound healing and tissue fibrosis. The main feature of EMyT is the activation of a myogenic program, leading to the induction of the α-smooth-muscle actin (SMA) gene in the transitioning epithelium. Recent research suggests that intercellular contacts are not merely passive targets, but are active contributors to EMT/EMyT. Indeed, our group showed previously that contact uncoupling or injury is necessary for TGFβ to induce EMyT (two-hit paradigm). Further, our previous work also revealed that Smad3, the main TGFβ-regulated transcription factor, binds to the Myocardin Related Transcription Factor (MRTF), the prime driver of SMA promoter, and inhibits MRTF’s transcriptional activity. During EMyT, Smad3 eventually degrades, which liberates the MRTF-driven myogenic program. However the mechanisms whereby cell contacts regulate the fate of Smad3 and MRTF during EMyT are poorly understood. Accordingly, the central aim of my studies was to explore the role of intercellular contacts, in particular that of Adherens Junction (AJs) in the induction of the myogenic reprogramming of the injured epithelium. This thesis describes two novel molecular mechanisms through which AJs impact EMyT. In the first part, we show β-catenin, an AJs component and transcriptional co-activator counteracts the inhibitory action of Smad3 on MRTF. Moreover we reveal that β-catenin is necessary to maintain MRTF stability via protecting MRTF from proteasomal degradation. Thus, β-catenin is an indispensable permissive factor for SMA expression. In the second part, we demonstrate that contact injury and TGFβ suppress the expression of the phosphatase PTEN. EMyT-related reduction or absence of PTEN potentiates Smad3 degradation. EMyT is associated with enhanced phosphorylation of the T179 residue in Smad3 linker region, and this event is necessary for Smad3 degradation. PTEN silencing increases the stimulatory effect of contact uncoupling and TGFβ on SMA promoter activity and SMA protein expression. Thus, the integrity of intercellular contacts regulates the level of PTEN, which in turn controls Smad3 stability through impacting on T179 phosphorylation. This new knowledge holds promises for targeted therapies and more effective prevention of the currently incurable fibroproliferative and fibrocontractile diseases.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [72]-87).

The post-natal regenerative capacity of skeletal muscle is attributed to myogenic satellite cells, which function as lineage-committed precursors to replace terminally differentiated muscle. The development and differentiation of the satellite cell lineage is regulated by Pax7 and the myogenic regulatory factors. While the expression of Pax7 is vital to the function of the satellite cell compartment, the paired domain alternative splicing events that regulate its DNA binding potential remain elusive. Interestingly, the generation of Pax7 splice variants differentially regulate Myf5 expression. We performed a global analysis of two Pax7 isoforms, which differ by a glycine-leucine dipeptide, to determine how paired domain splicing events modify the ability of Pax7 to regulate target genes. To this end, we observe that although the homeodomain is important for Pax7 binding, these isoform differences in the paired domain can regulate Pax7 targets during myogenesis. In addition to further examining the role of Pax7 during satellite cell proliferation and maintenance, it remains important to understand their downstream differentiation potential. Since activation of the canonical Wnt signalling pathway results in reduced regenerative efficiency in vivo, we undertook a global analysis of satellite cell derived myoblasts to examine their ability to respond to canonical Wnt signalling. We demonstrate that Wnt/β-Catenin signalling drives myogenic differentiation, via the myogenin-dependent control of follistatin expression, further fine-tuning the myogenic differentiation process. The effects of canonical Wnt signalling on myogenic differentiation complement our observations regarding Pax7 alternative splicing during myoblast proliferation and provide a greater comprehensive understanding of the molecular regulation of satellite cell development and differentiation during adult myogenesis.

Lysosomal storage diseases (LSDs) are metabolic diseases which occur as a result of a deficiency of one of the essential lysosomal enzymes, called glycohydrolases. A mutation in the gene encoding one of these enzymes leads to an accumulation of unwanted substrates, resulting in a variety of clinical manifestations. A common symptom found in LSDs is skeletal muscle dysfunction, which includes muscle weakness, atrophy and loss of muscle mass. The genes for lysosomal hydrolases are well characterized; however, much less is known about how mutations in these genes affect the cell and lead to the muscle dysfunction observed. One pathway of interest is autophagy; it has been shown to be essential for maintenance of skeletal muscles. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of Sialidosis, Sandhoff Disease, and GM1-Gangliosidosis in order to assess autophagy and its impact on myogenic differentiation in skeletal muscles. Our results demonstrated that autophagy is induced upstream (ULK1 phosphorylation) but is inhibited at the autophagosome to lysosome fusion (p62 upregulation) in LSDs. We also found that myoblast fusion and myogenic differentiation are impaired. We conclude that blocking autophagy impairs myogenic differentiation, which potentiates the muscle dysfunction observed in LSDs. This work highlights autophagy as a new pathway of interest and possible therapeutic target to alleviate muscle dysfunction in LSDs, and other similar neurodegenerative diseases. / Thesis / Master of Science (MSc) / Lysosomal storage diseases (LSDs) occur because of a deficiency of lysosomal glycohydrolases. A common symptom found in LSDs is skeletal muscle dysfunction. Little is known about how a deficiency of these enzymes leads to the clinical manifestations observed. However, one pathway of interest is autophagy. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of LSDs to assess autophagy and its impact on myogenic differentiation in skeletal muscles. We demonstrated that autophagy is induced and blocked, and that myoblast fusion and myogenic differentiation is impaired. We concluded that the induction and block of autophagy impairs myogenic differentiation, which potentiates muscle dysfunction.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 96-110.

No description available.

In normal tissues, blood supply is closely matched to tissue demand for wide ranges of oxygen demand and arterial pressure. This suggests that multiple mechanisms regulate blood flow. Theoretical models can be used to analyze these interacting mechanisms. One proposed mechanism for metabolic flow regulation involves the saturation-dependent release of ATP by red blood cells, which triggers an upstream conducted response signal and arteriolar vasodilation. To analyze this mechanism, oxygen and ATP levels are calculated along a flow pathway of seven representative segments, including two vasoactive arteriolar segments. The conducted response signal is dependent on ATP concentration. Arteriolar tone depends on the conducted response signal, local wall shear stress and wall tension. Arteriolar diameters are calculated based on vascular smooth muscle mechanics. The model can account for increases in perfusion consistent with experimental findings at low and moderate oxygen consumption rates despite the opposing effects of the myogenic and shear-dependent responses. Autoregulation, the maintenance of nearly constant blood flow as arterial pressure varies, is assessed in the presence or absence of the myogenic, shear-dependent and/or metabolic responses. The model results indicate that the combined effects of myogenic and metabolic regulation overcome the vasodilatory effect of the shear-dependent response to generate autoregulatory behavior. Capillary recruitment has been shown to increase the capacity for oxygen delivery during exercise. In the model, capillary density is assumed to depend on small arteriole diameter. The model predicts a significant increase in the range over which perfusion can be regulated when recruitment is included. Oscillations in diameter and tone are predicted under certain conditions, suggesting a novel mechanism for vasomotion. The conditions that give rise to oscillations are analyzed. It is shown that the appearance of oscillations depends in a complex way on a number of system parameters. In summary, the theoretical model provides a quantitative assessment of the myogenic, shear-dependent and metabolic responses that affect blood flow regulation and identifies a role for capillary recruitment and vasomotion in the control of blood flow.