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I am looking for Graduate students seeking a PhD only or post-doctoral fellows within 3 years of defending their PhD. Trainees will be actively involved in the translational research effort pertaining to understanding the physiological and/or pathophysiological role(s) of the serine protease family, granzymes, in the context of skin or vascular inflammation regeneration, in aging, autoimmune and/or chronic disease. Granzymes are a family of 5 serine proteases in humans. With the exception of GzmA and GzmB, very little is known about the other 3 proteases so there are many opportunities to carve out a niche, drug development, and for publications/patents. The job is highly translational and well suited for trainees seeking greater involvement in all steps in taking basic bench research discoveries through to clinical application and potentially commercialization. Our research program spans from basic biochemistry/molecular biology through to target validation, proof-of-concept in animal and human models with a strong connection to industry. Trainees will be responsible for designing and implementing studies to further elucidate mechanisms of disease through the use of in vitro and in vivo models. Dr. Granville’s laboratory (www.granzymes.com) utilizes a variety of disease models related to aging, autoimmunity, injury, inflammation and impaired healing as they pertain to cardiovascular, pulmonary, skin, musculoskeletal, and neuroinflammatory disorders. We also interact heavily with clinicians.
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
Myocarditis, inflammation of the heart muscle, is a spectrum of conditions causing significant morbidity and mortality, yet scientific and clinical knowledge related to this entity is limited. One of the most common and best studied causes of myocarditis is infection by coxsackievirus B3 (CVB3). An improved understanding of the science behind CVB3 myocarditis is critical to establishing better diagnostic and therapeutic strategies for affected individuals. CVB3 infection redirects numerous cellular pathways from physiologic processes to viral replication, often mediated by viral proteases. Two viral targets in this process are death associated protein 5 (DAP5) and nuclear pore complex protein 98 (Nup98). DAP5 is a translation initiation factor specific to internal ribosome entry site (IRES) mediated translation. Nup98 is a component of the nuclear pore complex and a transcription factor.In this thesis, I hypothesize that viral proteases contribute to the pathogenesis of viral myocarditis through interaction with DAP5 and Nup98, redirecting translation and transcription towards viral replication.Using in vitro (plasmid expressed viral proteases), in situ (CVB3 infection in cell culture), and in vivo (mouse myocarditis model) models, I demonstrate that viral protease 2A is responsible for the cleavage of DAP5 and Nup98 during CVB3 infection. Both cleavage events I show to be integral to the viral lifecycle using over expression of recombinant fragments and siRNA inhibition of that expression.These results suggest two previously unidentified targets for improved diagnostics and therapeutics for myocarditis, both areas for future research.
Background: Abdominal aortic aneurysm (AAA) is an age-related disease characterized by progressivedegradation of elastic lamellae, defective collagen architecture and medial smooth muscle cell loss. We previously demonstrated that knocking out the serine protease granzyme B (GZMB) reduces incidenceand severity of AAA in mice; however, while GZMB is known for its role in apoptosis, it also accumulates extracellularly during inflammation and can cleave extracellular matrix (ECM) components such as decorin and fibrillin-1. We hypothesized that GZMB contributes to AAA development through thedegradation of vascular ECM and that the inhibition of extracellular GZMB would reduce the incidence and severity of AAA progression.Methods: Human aneurysmal samples were obtained and apolipoprotein E(apoE)-knockout (KO),GZMB/apoE-double knockout (GDKO) and perforin/apoE-DKO (PDKO) mice were implanted withosmotic minipumps releasing angiotensin II for 28 days to induce AAA formation. Additional apoE-KOmice were injected with the GZMB inhibitor, serpin A3N (SA3N, 4-120 μg/kg) or anti-GZMB neutralizingantibody (1 mg/kg) prior to pump implantation. Tissues were assessed for aneurysm pathology,inflammation and ECM composition. Collagen content was analysed by second harmonic generation andtransmission electron microscopy.Results: Human aneurysmal tissues showed elevated levels of GZMB immunopositivity compared tocontrols. A significant reduction in AAA incidence and severity was observed in GDKO mice compared toapoE-KO, whereas perforin deficiency was not protective against AAA. A dose-dependent reduction inthe frequency of aortic rupture was observed in mice that received SA3N or anti-GZMB antibodytreatment. Pre-incubation with SA3N prevented decorin cleavage by GZMB in vitro. Reduced GZMB anda corresponding reduction in loss of adventitial decorin were observed in SA3N and anti-GZMB-treatedmice while collagen density was increased. Adventitial collagen from SA3N-treated mice exhibitedsignificantly higher fibre density and reduced fibril size irregularity.Conclusions: GZMB promotes destruction of the elastic lamellae via degradation of fibrillin-1 anddestabilization of elastic microfibrils while GZMB-mediated degradation of decorin contributes to loss ofadventitial collagen organization and density. The extracellular inhibition of GZMB prevented decorinloss and enabled a beneficial remodelling of adventitial collagen in response to medial injury, leading tohigher vessel tensile strength and increased resistance to aortic rupture.
Granzyme B (GzmB) is a serine protease that can be released into the extracellular spacesby immune cells during chronic inflammation where it is capable of degrading severalcomponents of the extracellular matrix (ECM). Several chronic inflammatory skin diseases havedemonstrated elevated levels of GzmB however the exact role of GzmB in the skin remainspoorly understood. Apolipoprotein E (ApoE) is a protein highly expressed in the skin, where itcan regulate inflammation through its anti-oxidative and anti-inflammatory properties. Micedeficient in ApoE develop an inflammatory skin phenotype when fed a high fat diet indicative ofpremature aging featuring ECM remodeling, hair graying, hair loss and frailty. I thereforehypothesized that GzmB contributes to skin aging, injury and impaired healing in ApoEknockout (ApoE-KO) mice through the degradation of ECM proteins. In the present dissertation,I identified the high fat diet-fed ApoE-KO mouse as a model that displays several characteristicfeatures of skin aging including skin thinning and collagen disorganization. Furtherinvestigations also identified that high fat diet-fed ApoE-KO mice show defects in cutaneouswound healing such as delayed wound closure, reduced contraction and altered collagen content.These changes became worse with age and high fat diet. To test the role of GzmB in this process,GzmB/ApoE double knockout (DKO) mice were generated. These DKO mice were protectedfrom skin thinning and collagen disorganization even when fed a high fat diet, suggesting thatGzmB plays a role in ECM remodeling during aging of the skin in ApoE-KO mice. Furtherinvestigation revealed that GzmB-mediated degradation of the proteoglycan decorin is likely tobe a key mechanism by which GzmB contributes to collagen disorganization and skin aging inApoE-KO mice. Furthermore, DKO mice showed improved wound healing compared to ApoEKOmice featuring faster wound closure, increased contraction and reduced fibronectin degradation. In vitro cleavage assays revealed that fibronectin fragments generated by GzmBmatched those identified in non-healing ApoE-KO mouse wounds. In summary, my findingssuggest that extracellular GzmB contributes to skin aging and impaired healing in ApoE-KOmouse skin through the degradation of ECM components such as decorin and fibronectin.
Granzyme B (GZMB) is a serine protease that is expressed by a variety of immune cells and is abundant in a large number of chronic inflammatory disorders. GZMB is highly expressed in cytotoxic lymphocytes where it serves as the main effector molecule of the granule exocytosis pathway by which cytotoxic immune cells mediate target cell death through intracellular delivery of GZMB, leading to activation of apoptotic signaling cascades. GZMB can also accumulate extracellularly during inflammation, where it can cleave a range of extracellular matrix (ECM) proteins that may disrupt cell-matrix interactions and modulate the bioavailability of matrix-bound growth factors. In this dissertation I have explored the intracellular and extracellular roles of GZMB in vascular remodeling in disease. By examining human atherosclerotic plaques, I discovered an imbalance between GZMB and its endogenous inhibitor, proteinase inhibitor 9 (PI-9). PI-9 expression by vascular smooth muscle cells (VSMC) in plaques was reduced with increased disease severity. Elevated levels of GZMB in advanced lesions were correlated with reduced PI-9 expression and increased VSMC apoptosis. These findings suggest that VSMC are more susceptible to GZMB-induced apoptosis in advanced lesions due to reduced PI-9 expression. While examining the extracellular activities of GZMB on vascular remodeling, I focused on the role of GZMB-mediated cleavage of fibronectin (FN), a known GZMB substrate. FN has a major role in regulating angiogenesis as it facilitates endothelial cell (EC) migration and capillary formation, as well as binding to angiogenic growth factors in the ECM including vascular endothelial growth factor (VEGF). VEGF is a potent vascular permeabilizing agent that is sequestered in the ECM by binding FN. GZMB-mediated FN cleavage resulted in reduced EC adhesion, migration and capillary tube formation. In addition, GZMB-mediated FN cleavage induced the release of VEGF from the ECM and promoted VEGF-dependent vascular leakage in vivo. Thus, GZMB may contribute to the progression and/or persistence of chronic inflammation by dysregulating angiogenesis and promoting vascular permeability. Collectively, the results of this work suggest that both intracellular and extracellular GZMB activities contribute to vascular remodeling and pathological angiogenesis.
Granzyme B (GzmB) is a serine protease that contributes to immune-mediated elimination ofcells by initiating a tightly-regulated form of death known as apoptosis. However, duringinflammation, GzmB leaks out and accumulates in the extracellular space, retains its activity,and proficiently cleaves extracellular matrix (ECM) proteins. I therefore hypothesized thatextracellular GzmB is capable of cleaving novel ECM substrates, contributing to dysregulatedECM integrity and function in disease. In the present dissertation I identified eleven novelextracellular GzmB substrates. Further investigations revealed that GzmB-mediatedproteoglycan cleavage was implicated in the dysregulation of active transforming growth factorbeta(TGF-β) sequestration and bioavailability. GzmB cleavage sites were identified in biglycanand betaglycan and active TGF-β was shown to be released from decorin, biglycan andbetaglycan. The pathophysiological role of my findings were further investigated and validatedusing animal models of disesase in which inflammation and elevated GzmB are observed.Evidence of fibrillin-1 and decorin cleavage were observed in atherosclerosis, abdominal aorticaneurysm and in skin aging pathogenesis. I also assessed the activity of GzmB in advancedatherosclerosis using perforin/apolipoprotein E- double knockout (Perf/apoE-DKO) andgranzyme B/apolipoprotein E-double knockout (GzmB/apoE-DKO) mice. Interestingly, unlikeour aneurysm findings whereby only GzmB/apoE-DKO mice were protected, both Perf/apoEDKOand GzmB/apoE-DKO mice were protected from atherosclerosis compared to apoE-KOcontrols, suggesting the intracellular Perf-dependent activities of granzymes are also importantin the pathogenesis of atherosclerosis. In summary, GzmB is a protease that functions bothintracellularly and extracellularly in disease. My findings suggest that the use of Perf knockoutmice alone to study the role of GzmB in disease should be re-evaluated given the increasing evidence in both animal models and in human disease showing elevated GzmB in bodily fluids is associated with inflammation and age.
Master's Student Supervision (2010 - 2018)
The skin is comprised of multiple layers of keratinocytes which together form a barrier to the external environment, regulating temperature, water loss, and pathogen exposure. As such the skin barrier is vital for health as well as disease prevention. Disruption of the epithelial barrier can result in infection, allergen exposure, and inflammation, culminating into severe conditions. Many autoimmune conditions, such as pemphigus, involve a dysregulation and accumulation of immune cells, this results in a disruption in skin barrier causing a loss of function. Granzyme B (GzmB) is a serine protease that is expressed and secreted by a variety of immune and non-immune cells. It can accumulate in the extracellular milieu and retain its proteolytic functions resulting in chronic inflammation and impaired tissue repair due to extracellular matrix (ECM) remodeling. As such, I hypothesized that GzmB disrupts epithelial barrier function through the proteolytic cleavage of cell junction proteins. The present study investigated the impact of GzmB on epithelial barrier dysfunction using Electric Cell-substrate Impedance Sensing (ECIS) and western blot analyses of intercellular junction cleavage fragments. Human formalin fixed, paraffin embedded blistered skin tissue was assessed for the presence of GzmB. GzmB treatment resulted in a loss of E-cadherin staining on the cell membrane which was supported by western blot analysis of the cell supernatants. Additionally, we observed a dose-dependent increase in E-cadherin fragmentation in GzmB-treated cells compared to controls. HaCaT cells exhibited a significant decrease in barrier function when treated with GzmB while cells treated with GzmB in the presence of a specific GzmB inhibitor remained unaffected. While absent in normal skin, GzmB was observed in abundance within the intra-epidermal blister in addition to the surrounding epithelium. In summary, GzmB contributes to a decline in epithelial barrier function in part through the proteolytic cleavage of cell-cell junctions.
No abstract available.
Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte-induced target cell death. However, non-lethal roles for granzymes, including Granzyme K (GzK), have been recently proposed. As recent studies have observed elevated levels of GzK in plasma of patients diagnosed with sepsis, we hypothesized that extracellular GzK induces a pro-inflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated Protease Activated Receptor-1 (PAR-1) leading to increased IL-6 and MCP-1 production in Human Umbilical Venous Endothelial Cells (HUVEC). Enhanced expression of ICAM-1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the MAPK p38 pathway for ICAM-1 expression, and both the p38 and the ERK1/2 pathways in cytokine production. GzK also increased TNFα–induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, IαIp, significantly inhibited GzK activity in vitro. In summary, extracellular GzK is not cytotoxic but promotes a pro-inflammatory response in endothelial cells.
Chronic, non-healing wounds are a major complication of diabetes and are characterized by chronic inflammation and excessive protease activity. While once thought to function primarily as a pro-apoptotic serine protease, granzyme B (GzmB) can also accumulate in the extracellular matrix during chronic inflammation and cleave extracellular matrix (ECM) proteins that are essential for proper wound healing, including fibronectin. We hypothesized that GzmB contributes to the pathogenesis of impaired diabetic wound healing through excessive degradation of the ECM. In the first part of the thesis, we demonstrated that the majority of GzmB was secreted by mast cells and localized in the wound edges and granulation tissues of completely reepithelialized diabetic mouse wounds at higher levels. Subsequently, we observed that GzmB induced detachment of mouse embryonic fibroblasts and also showed that co-incubation with a mouse serine protease inhibitor, serpina3n (SA3N), abrogated this effect. Finally, we administered SA3N to diabetic mouse wounds and found that wound closure including both reepithelialization and contraction were significantly increased in wounds treated with SA3N. Histological and immunohistochemical analyses of the SA3N-treated wounds revealed a more mature, proliferative granulation tissue phenotype as indicated by increased cells with proliferative activity, vascularization, contractile myofibroblasts, as well as collagen deposition in remodeling tissues. Skin homogenates from SA3N-treated wounds also exhibited greater levels of full-length intact fibronectin when compared to control wounds. In summary, our findings suggested that GzmB contributes to the pathogenesis of diabetic wound healing through the proteolytic cleavage of fibronectin that are essential for normal wound closure, and that inhibition of GzmB can promote granulation tissue maturation and collagen deposition. These results offer preliminary evidence that a GzmB inhibitor may be a relevant therapeutic target in wound management therapy.