Tetralogy of Fallot (TOF) is a form of complex congenital heart disease (CHD) with clinical and genetic heterogeneity. Of the few known causes, 22q11.2 deletion syndrome is most common. We sought to define other clinical subgroups by focusing on congenital cardiac and extra-cardiac features, and cardiac outcome. Patients were prospectively categorized as “syndromic” if they had at least two of three features: dysmorphic facies, learning difficulties or voice abnormalities. We compared cardiac and extra-cardiac characteristics, and late cardiac outcomes between the syndromic group and a nonsyndromic control group. The syndromic group had a more complex cardiac disease, was at elevated risk for developing later onset conditions including neuropsychiatric disorders, endocrine disorders, and hearing deficits, and had a higher mortality rate compared to the nonsyndromic group. Increased awareness of this subgroup with a multisystem condition may be helpful for optimizing management and identifying individuals for referral to medical genetics.

Tetralogy of Fallot (TOF) is a form of complex congenital heart disease (CHD) with clinical and genetic heterogeneity. Of the few known causes, 22q11.2 deletion syndrome is most common. We sought to define other clinical subgroups by focusing on congenital cardiac and extra-cardiac features, and cardiac outcome. Patients were prospectively categorized as “syndromic” if they had at least two of three features: dysmorphic facies, learning difficulties or voice abnormalities. We compared cardiac and extra-cardiac characteristics, and late cardiac outcomes between the syndromic group and a nonsyndromic control group. The syndromic group had a more complex cardiac disease, was at elevated risk for developing later onset conditions including neuropsychiatric disorders, endocrine disorders, and hearing deficits, and had a higher mortality rate compared to the nonsyndromic group. Increased awareness of this subgroup with a multisystem condition may be helpful for optimizing management and identifying individuals for referral to medical genetics.

Background: Total joint arthroplasty (TJA) is indicated in persons with end-stage arthritis of the hip and knee (THA and TKA, respectively). While most TJAs are performed for osteoarthritis (OA), 3%-13% are performed in patients with rheumatoid arthritis (RA). Most of the evidence regarding complications following TJA is based on studies of patients with OA, with little known about recipients with RA. The purpose of this thesis was to summarize current evidence on the rates of complications following TJA in patients with RA, to quantify this risk using validated methods, and to determine the impact of surgeon experience performing TJA in persons with RA on this risk. Methods: For reports published between 1990 and 2011, we evaluated the evidence regarding the risk of complications following TJA in RA using qualitative and quantitative methods. In a cohort of recipients of primary elective THA or TKA between 2002 and 2009, in Ontario, Canada, we identified patients with RA using a validated algorithm. Multivariable Cox proportional hazards regression was used to evaluate the relationship between arthritis type (RA, OA, other) and the occurrence of surgical complications, and to evaluate the impact of surgeon experience on the risk of a complication. Results: Forty published studies were reviewed. Relative to TJA recipients with OA, those with RA were at increased risk of dislocation following THA, and increased risk for joint infection following TKA. These findings were confirmed in our cohort study: adjusted hazard ratio (HR) for dislocation 1.91, p=0.001; adjusted HR for infection 1.47, p=0.03). In TJA recipients with RA, greater surgeon RA volume was associated with a reduced risk for surgical complications (adjusted HR per 10 additional cases: 0.81, p=0.002). Conclusions: In a population-based cohort of primary elective TJA recipients, patients with RA were at significantly increased risk for dislocation following THA and joint infection following TKA. Increased surgeon experience performing TJA in patients with RA attenuated the risk for surgical complications among TJA recipients with RA. Further research is required to identify the mediators of the increased complication risk in patients with RA, and to delineate strategies to optimize outcomes in these patients.

Elastic fibers are major components of ECM, providing tissues with resilience and elastic recoil. They consist of the insoluble elastin made of cross-linked precursor tropoelastin monomers and the microfibrillar scaffold. Various connective tissue disorders have been linked to elastic fiber malformation. In addition, it has been shown that decreased vascular elastin contents correlate with the rapid progression of atherosclerosis in patients with diabetes mellitus. However, it is unknown whether insulin can directly modulate elastogenesis. This question was addressed in this thesis. Our study revealed that insulin stimulated elastogenesis in cultures of human aortic SMCs and skin fibroblasts, and that this effect of insulin was transduced through the insulin receptor. We found that insulin might initiate elastin gene expression and enhance tropoelastin secretion. We also presented novel preliminary data indicating that Glut10 might bind to a fraction of intracellular tropoelastin, and that insulin might modulate the association between these two proteins.

Elastic fibers are major components of ECM, providing tissues with resilience and elastic recoil. They consist of the insoluble elastin made of cross-linked precursor tropoelastin monomers and the microfibrillar scaffold. Various connective tissue disorders have been linked to elastic fiber malformation. In addition, it has been shown that decreased vascular elastin contents correlate with the rapid progression of atherosclerosis in patients with diabetes mellitus. However, it is unknown whether insulin can directly modulate elastogenesis. This question was addressed in this thesis. Our study revealed that insulin stimulated elastogenesis in cultures of human aortic SMCs and skin fibroblasts, and that this effect of insulin was transduced through the insulin receptor. We found that insulin might initiate elastin gene expression and enhance tropoelastin secretion. We also presented novel preliminary data indicating that Glut10 might bind to a fraction of intracellular tropoelastin, and that insulin might modulate the association between these two proteins.

Background: Total joint arthroplasty (TJA) is indicated in persons with end-stage arthritis of the hip and knee (THA and TKA, respectively). While most TJAs are performed for osteoarthritis (OA), 3%-13% are performed in patients with rheumatoid arthritis (RA). Most of the evidence regarding complications following TJA is based on studies of patients with OA, with little known about recipients with RA. The purpose of this thesis was to summarize current evidence on the rates of complications following TJA in patients with RA, to quantify this risk using validated methods, and to determine the impact of surgeon experience performing TJA in persons with RA on this risk. Methods: For reports published between 1990 and 2011, we evaluated the evidence regarding the risk of complications following TJA in RA using qualitative and quantitative methods. In a cohort of recipients of primary elective THA or TKA between 2002 and 2009, in Ontario, Canada, we identified patients with RA using a validated algorithm. Multivariable Cox proportional hazards regression was used to evaluate the relationship between arthritis type (RA, OA, other) and the occurrence of surgical complications, and to evaluate the impact of surgeon experience on the risk of a complication. Results: Forty published studies were reviewed. Relative to TJA recipients with OA, those with RA were at increased risk of dislocation following THA, and increased risk for joint infection following TKA. These findings were confirmed in our cohort study: adjusted hazard ratio (HR) for dislocation 1.91, p=0.001; adjusted HR for infection 1.47, p=0.03). In TJA recipients with RA, greater surgeon RA volume was associated with a reduced risk for surgical complications (adjusted HR per 10 additional cases: 0.81, p=0.002). Conclusions: In a population-based cohort of primary elective TJA recipients, patients with RA were at significantly increased risk for dislocation following THA and joint infection following TKA. Increased surgeon experience performing TJA in patients with RA attenuated the risk for surgical complications among TJA recipients with RA. Further research is required to identify the mediators of the increased complication risk in patients with RA, and to delineate strategies to optimize outcomes in these patients.

We investigated the capacity of human umbilical cord perivascular cells (HUCPVCs) to repair tendon damage in a collagenase tendon injury model in rats. Our hypothesis was that HUCPVCs would be able to produce an organized extracellular matrix and facilitate tissue regeneration in this rat model. HUCPVCs were injected, in collagenase, into the Achilles tendon of nude rats. Harvested tendons showed the presence of HUCPVCs at the site of injury, whose morphology changed from rounded to elongated over the post-injury time of 30 days. Human genes for collagen type 1 and -actin were expressed at all time points and there was also a signi cant increase in the tensile strength and sti ness values at 30 day post-injury in the experimental group. We conclude that HUCPVCs facilitated regeneration in our model, and thus may be a putative cell source for the treatment of tendon and ligament injuries.

We investigated the capacity of human umbilical cord perivascular cells (HUCPVCs) to repair tendon damage in a collagenase tendon injury model in rats. Our hypothesis was that HUCPVCs would be able to produce an organized extracellular matrix and facilitate tissue regeneration in this rat model. HUCPVCs were injected, in collagenase, into the Achilles tendon of nude rats. Harvested tendons showed the presence of HUCPVCs at the site of injury, whose morphology changed from rounded to elongated over the post-injury time of 30 days. Human genes for collagen type 1 and -actin were expressed at all time points and there was also a signi cant increase in the tensile strength and sti ness values at 30 day post-injury in the experimental group. We conclude that HUCPVCs facilitated regeneration in our model, and thus may be a putative cell source for the treatment of tendon and ligament injuries.

Juvenile GM2 gangliosidosis (jGM2) is a group of inherited neurodegenerative diseases caused by deficiency of lysosomal β-hexosaminidase A (Hex A) resulting in GM2 ganglioside accumulation in brain. Like many other lysosomal storage diseases (LSDs), no specific treatment currently exists. In order to establish clinical outcomes for the investigation of potential therapies for jGM2, I collected comprehensive information on the natural history of the condition by studying retrospective and prospectively a cohort of 21 patients with the disease, and reviewing previously published reports of 134 patients. Several symptoms at disease onset, symptom latencies, and the survival curve were described. Genotype-phenotype correlations and neuroradiological findings were also studied. Based on pre-clinical results in animal models, we studied substrate reduction therapy (SRT), with miglustat, in a phase I/II clinical trial to assess its pharmacokinetics (PK), safety, tolerability in infantile and jGM2. Miglustat showed a PK profile similar to the one found in adult patients. The drug was found to be safe and well-tolerated in patients with jGM2, with diarrhea and weight loss being the most common drug-related adverse events. The analysis of efficacy showed that SRT was unable to arrest the full neurological progression of the condition; however, relative stabilization of cognitive function was noted, which was consistent with brain MRI findings. Because of the limited efficacy obtained with SRT, enzyme-enhancement therapy was considered to be an attractive alternative therapy for the late onset forms of GM2 gangliosidosis. Screening of a FDA-approved library of approved therapeutic compounds resulted in the identification of pyrimethamine, as a potential pharmacological chaperone for mutant forms of Hex A. Relative enhancements of enzyme activity and protein levels were observed in patient cells treated with therapeutic concentrations of drug. Applying the same principles, ambroxol was identified as a potential PC for mutant glucocerebrosidase (GCC), the lysosomal enzyme that when deficient causes Gaucher disease (GD). Significant increases of residual mutant GCC were observed in cultured patients cells with type 1 GD. In conclusion, principles developed in the course of studies on jGM2 were shown to be useful for the investigation of novel small-molecule therapies for LSDs, associated with significant neurodegeneration.

Diabetic nephropathy is the leading cause of end-stage renal disease, yet the mechanisms responsible for hyperglycemia-induced kidney injury have not been fully elucidated. Activation of the renin-angiotensin system and NADPH oxidase-dependent generation of reactive oxygen species are important mediators of chronic kidney disease. I first studied the effect of ACE2, an important enzyme in the renin-angiotensin system, in diabetic kidney injury in the Akita mouse and related the effect to angiotensin peptide and NADPH oxidase. I then demonstrated the interaction between Angiotensin II, the main substrate, and angiotensin-(1-7), the main product of ACE2, respectively, on cell signaling in mesangial cells to better understand the in vitro effect of ACE2. Finally I studied the effect of deletion of p47phox, a regulatory subunit of the NADPH oxidase, on initiation and progression of diabetic nephropathy in the Akita mouse and mesangial cell. Administration of human recombinant ACE2 decreased angiotensin II levels, increased angiotensin-(1-7) levels, normalized NADPH oxidase activity in the Akita mice, and ameliorated diabetes-induced kidney injury. In vitro, hrACE2 attenuated both high glucose and ANG II–induced oxidative stress and NADPH oxidase activity in mesangial cells. Ang-(1–7)-induced ERK1/2 phosphorylation in mesangial cells in a mas receptor-cAMP-PKA-dependent manner. This effect of ang-(1-7) on ERK1/2 phosphorylation is not mediated by AT1R, AT2R, epidermal growth factor or NADPH oxidase. Pre-treatment with Ang-(1-7) attenuated Ang II-induced NADPH oxidase activity and ERK1/2 activation also in a cAMP-PKA-dependent manner. Deletion of p47phox not only reduced diabetes-induced kidney injury but also reduced hyperglycemia by increasing pancreatic and circulating insulin concentrations. p47phox-/- mice exhibited improved glucose tolerance but modestly decreased insulin sensitivity. Deletion of p47phox attenuated high glucose-induced activation of NADPH oxidase and pro-fibrotic gene expression in mesangial cells. There was a positive correlation between p47phox and collagen Iα1 mRNA levels in renal biopsy samples from control subjects and subjects with diabetic nephropathy. The data generated in this thesis strongly suggest a protective role of ACE2, via Ang-(1-7), and a deleterious role of p47phox in diabetic nephropathy. Future therapeutic strategies should include enhancing ACE2 activity in the kidney and inhibiting p47phox-dependent activation of NADPH oxidase in both the kidney and the pancreas.