The domestic dog, Canis lupus familiaris, plays many roles in the lives of humans. Additionally, the dog is recognized for its potential as a model for many human hereditary diseases. Thus, the genetics and genomics of the dog are being studied extensively in order to facilitate its use as a model, as well as to help the dog for its own sake. As part of this research effort, our laboratory has added type I markers (i.e., the acidic and basic keratins, c-kit, type I and IV collagens, and the gene encoding uromodulin) to the emerging map of the canine genome. The mapping of genes, particularly those in large gene families such as the collagens, is valuable because it rapidly increases the density of gene loci on the map and provides insight regarding conservation of synteny between the dog and other mammals. The major focus of work reported here is the genetics of X-linked Alport syndrome (XLAS), a terminal renal disease that affects the human and the dog. The disease results from mutations in COL4A5, a type IV collagen gene. Reported here are the 1) sequencing and mapping of the canine cDNA encoding uromodulin, 2) mapping of the type I and type IV collagen genes, 3) sequencing of the full-length cDNA of canine COL4A5, 4) identification of a 10 bp deletion in COL4A5, causative for XLAS in our colony of mixed breed dogs, 5) development of a genetic test for identification of affected and carrier dogs in the colony and 6) assessment of gene expression in the kidneys of normal and XLAS-dogs. This assessment was performed using a canine-specific oligonucleotide microarray. XLAS dogs demonstrated up-regulation of many genes involved in extracellular matrix reorganization, cell structure, and immune response, as expected in a glomerulopathy with tubulointerstitial nephritis. Trends were verified by quantitative RT-PCR. A review of the current status of canine genetics research, and current understanding of hereditary diseases in the dog, concludes this dissertation.

Design of self-assembling ABC-type collagen triple helical heterotrimers is challenging due to the number of competing species that can be formed in ternary mixture of peptides with a high propensity to fold into triple helices and the fact that well understood rules for pair-wise amino acid stabilization of the canonical collagen triple helix have remained elusive. Given the required one amino acid stagger between adjacent peptide strands in this fold, a ternary mixture of peptides can form as many as 27 triple helices with unique composition or register. Previously we have demonstrated that electrostatic interactions can be used to bias the helix population towards a desired target but the presence of competing states in mixtures has remained an outstanding problem. In this work we use high-resolution structural biology techniques to do a detailed study of stabilizing pair-wise interactions between positively and negatively charged amino acids in triple helices. Two types of contacts with distinct sequence requirements depending on the relative stagger of the interacting chains are observed: axial and lateral. Such register-specific interactions are crucial for the understanding of the registration process of collagens and the overall stability of proteins in this family. Using this knowledge we developed distinct design strategies to improve the specificity of our designed systems towards the desired ABC heterotrimeric target state. We validate our strategies through the synthesis and characterization of the designed sequences and show that they self-assemble into a highly stable ABC triple helices with control over composition in the case of the rational approach and with control over both composition and register in the case of the computational approach.

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.

During atherosclerosis and restenosis, there is an extensive amount of collagen synthesis and degradation. Changes in the types of collagen present can have profound effects on vascular smooth muscle cell (SMC) proliferation and migration. Type VIII collagen, which is normally present at low levels within the mature vascular system, is greatly increased during atherogenesis. The central theme of this thesis is to determine the role of type VIII collagen in the pathogenesis of atherosclerosis and restenosis. In the first study, we demonstrated the importance of type VIII collagen in SMC migration and proliferation. SMCs from type VIII collagen-deficient mice display increased adhesion and decreased spreading, migration, and proliferation compared to SMCs from wild-type mice. Treatment of SMCs from type VIII collagen-deficient mice with exogenous type VIII collagen can rescue the defects. In the second study, we determined that type VIII collagen exerts its effects through regulation of MMP-2 expression. Type VIII collagen-deficient SMCs have decreased levels of MMP-2 and are impaired in chemotaxis toward PDGF-BB and in their ability to contract thick collagen gels. We found that decreasing endogenous MMP-2 levels in normal SMCs or adding exogenous collagen to type VIII collagen-deficient SMCs is sufficient to recapitulate the type VIII collagen-deficient or wild-type SMC phenotype, respectively. In the third study, we investigated the contribution of type VIII collagen to intimal hyperplasia after mechanical injury in the mouse. We found that type VIII collagen-deficient mice display a 35% reduction in intimal hyperplasia and attenuated vessel remodeling after femoral artery wire injury, establishing a role for type VIII collagen in restenosis. The results of the work presented in this thesis demonstrate that production of type VIII collagen confers an SMC phenotype with a greater propencity for proliferation and migration. These effects are in part mediated through regulation of MMP-2 expression and activation. We conclude that the increases in type VIII collagen production that occur during atherosclerosis and restenosis contribute to the capacity of SMCs to alter the existing extracellular matrix in a manner which permits enhanced migration.

This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.

Drug development research on wound repair is challenging and inefficient due to the complex nature of wound healing and scarring processes and the limitations of available in vitro or in vivo models used for preclinical drug testing. Many patients who undergo elective back surgery develop post-surgical complications resulting from excess peridural scarring in and around the site of operation. We tested the effects of two anti-inflammatory compounds, quercetin and L-2-oxothiazolidine-4-carboxylate (OTC), in ameliorating peridural scar formation following spinal laminectomy surgery in laboratory rats. Western blot and immunocytochemical analyses indicated that the peridural scar tissue contained MyoD-positive myoblast cells and expressed prolyl-4-hydroxylase (P4H), a fibroblast marker. Treatment with 1 mM OTC reduced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) at 21 days post-surgery suggesting potential anti-scarring mechanism. However, large animal to animal variation in the expression levels of collagen biosynthesis markers made it difficult to demonstrate any efficacy of quercetin or OTC in reducing peridural scar formation. The shortcomings of this live animal approach led us to develop a novel three-dimensional (3-D) <i>in vitro</i> wound repair model for evaluating quercetin and OTC effects. High-density micromass co-cultures seeded at a 1:3 ratio of FR 3T3 fibroblast cells and L8 myoblast cells formed 3-D microtissues <i>in vitro</i> that expressed MyoD, P4H, and á-smooth muscle actin. The micromass tissue layer remained adherent to the culture plate when inflicted with a single laceration injury, which allowed monitoring of cell migration into the wound site. Wounded cultures were treated with quercetin, OTC and other agents (TGF- â1, mitomycin, p38 inhibitor SB202190, ERK inhibitor PD184352) to determine their effects on collagen accumulation, wound closure rates, MAPK activation, and gene transcript expression. Both OTC and quercetin treatments reduced collagen biosynthesis in dose-dependent manner. In addition, 1.5 mM OTC accelerated wound closure and significantly reduced p38 MAPK activation without affecting ERK1/2. In contrast, 40 µM quercetin delayed wound closure in micromass co-cultures and reduced ERK1/2 activation. Our in vitro findings suggest that OTC might have potential as an anti-scarring agent. Importantly, our novel micromass co-culture system shows promise as an improved 3-D scaffold-free in vitro model for use in preclinical drug development research.

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.

During atherosclerosis and restenosis, there is an extensive amount of collagen synthesis and degradation. Changes in the types of collagen present can have profound effects on vascular smooth muscle cell (SMC) proliferation and migration. Type VIII collagen, which is normally present at low levels within the mature vascular system, is greatly increased during atherogenesis. The central theme of this thesis is to determine the role of type VIII collagen in the pathogenesis of atherosclerosis and restenosis. In the first study, we demonstrated the importance of type VIII collagen in SMC migration and proliferation. SMCs from type VIII collagen-deficient mice display increased adhesion and decreased spreading, migration, and proliferation compared to SMCs from wild-type mice. Treatment of SMCs from type VIII collagen-deficient mice with exogenous type VIII collagen can rescue the defects. In the second study, we determined that type VIII collagen exerts its effects through regulation of MMP-2 expression. Type VIII collagen-deficient SMCs have decreased levels of MMP-2 and are impaired in chemotaxis toward PDGF-BB and in their ability to contract thick collagen gels. We found that decreasing endogenous MMP-2 levels in normal SMCs or adding exogenous collagen to type VIII collagen-deficient SMCs is sufficient to recapitulate the type VIII collagen-deficient or wild-type SMC phenotype, respectively. In the third study, we investigated the contribution of type VIII collagen to intimal hyperplasia after mechanical injury in the mouse. We found that type VIII collagen-deficient mice display a 35% reduction in intimal hyperplasia and attenuated vessel remodeling after femoral artery wire injury, establishing a role for type VIII collagen in restenosis. The results of the work presented in this thesis demonstrate that production of type VIII collagen confers an SMC phenotype with a greater propencity for proliferation and migration. These effects are in part mediated through regulation of MMP-2 expression and activation. We conclude that the increases in type VIII collagen production that occur during atherosclerosis and restenosis contribute to the capacity of SMCs to alter the existing extracellular matrix in a manner which permits enhanced migration.

This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.

Drug development research on wound repair is challenging and inefficient due to the complex nature of wound healing and scarring processes and the limitations of available in vitro or in vivo models used for preclinical drug testing. Many patients who undergo elective back surgery develop post-surgical complications resulting from excess peridural scarring in and around the site of operation. We tested the effects of two anti-inflammatory compounds, quercetin and L-2-oxothiazolidine-4-carboxylate (OTC), in ameliorating peridural scar formation following spinal laminectomy surgery in laboratory rats. Western blot and immunocytochemical analyses indicated that the peridural scar tissue contained MyoD-positive myoblast cells and expressed prolyl-4-hydroxylase (P4H), a fibroblast marker. Treatment with 1 mM OTC reduced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) at 21 days post-surgery suggesting potential anti-scarring mechanism. However, large animal to animal variation in the expression levels of collagen biosynthesis markers made it difficult to demonstrate any efficacy of quercetin or OTC in reducing peridural scar formation. The shortcomings of this live animal approach led us to develop a novel three-dimensional (3-D) <i>in vitro</i> wound repair model for evaluating quercetin and OTC effects. High-density micromass co-cultures seeded at a 1:3 ratio of FR 3T3 fibroblast cells and L8 myoblast cells formed 3-D microtissues <i>in vitro</i> that expressed MyoD, P4H, and á-smooth muscle actin. The micromass tissue layer remained adherent to the culture plate when inflicted with a single laceration injury, which allowed monitoring of cell migration into the wound site. Wounded cultures were treated with quercetin, OTC and other agents (TGF- â1, mitomycin, p38 inhibitor SB202190, ERK inhibitor PD184352) to determine their effects on collagen accumulation, wound closure rates, MAPK activation, and gene transcript expression. Both OTC and quercetin treatments reduced collagen biosynthesis in dose-dependent manner. In addition, 1.5 mM OTC accelerated wound closure and significantly reduced p38 MAPK activation without affecting ERK1/2. In contrast, 40 µM quercetin delayed wound closure in micromass co-cultures and reduced ERK1/2 activation. Our in vitro findings suggest that OTC might have potential as an anti-scarring agent. Importantly, our novel micromass co-culture system shows promise as an improved 3-D scaffold-free in vitro model for use in preclinical drug development research.