Apoptosis-induced compensatory proliferation (AiP) is a mechanism that maintains tissue homeostasis after stress-induced cell death. During AiP, apoptotic cells induce proliferation of the neighboring surviving cells to compensate for tissue loss. AiP is important for wound healing and tissue regeneration in several model organisms. Additionally, AiP is an important feature of tumorigenesis and tumor relapse as it contributes to tumor repopulation following radiation or chemotherapy. Using an overgrowth tumor model (“undead tissue”) in Drosophila melanogaster, we determined that the initiator caspase Dronc promotes generation of extracellular Reactive Oxygen Species (ROS), which drive activation of the stress kinase JNK and downstream mitogens to promote AiP. We also observed increased numbers of Drosophila macrophages, termed hemocytes, which are attracted to undead tissue. However, the specific mechanisms by which macrophages are recruited to undead tissue are still unclear. Here, we report that the tissue microenvironment of the overgrown undead tissue directs macrophage recruitment during AiP. We demonstrate that ROS, JNK, and the matrix metalloproteinase Mmp2 are important for recruiting macrophages. Mechanistically, undead tissue-produced ROS and active JNK damage the basement membrane (BM) surrounding the undead tissue, by upregulating the expression and activity of Mmp2. The damaged BM then recruits macrophages to the undead tissue. Taken together, we propose a model in which the ROS-JNK-Mmp2 signaling axis damages the BM of undead tissue, resulting in changes in the tissue microenvironment that recruit macrophages to the area of damage to promote AiP and overgrowth.

Atherosclerosis is a macrophage-dominated nonresolving inflammatory disease of the arterial wall. Macrophage processes, including apoptosis, influence lesion development in atherosclerosis. Cannabinoids, compounds structurally related to Δ9-tetrahydrocannabinol (THC), the active ingredient in marijuana, exert their effects through cannabinoid receptors, CB1 and CB2. Cannabinoid treatment, THC or Win55,212-2, reduces atherosclerosis in ApoE-null mice by a mechanism thought to involve CB2. However, the exact role of CB2 in atherosclerosis remains unclear. We found that CB2-null macrophages are resistant to oxysterol/oxLDL-induced apoptosis leading us to hypothesize that CB2 may modulate macrophage apoptosis in atherosclerosis. To determine the functions of CB2 in atherosclerosis, we fed low density lipoprotein receptor-null (Ldlr-/-) and Ldlr-/- mice genetically deficient in CB2, an atherogenic diet for 8 and 12 weeks. CB2 deficiency did not significantly affect aortic root lesion area after 8 or 12 weeks; however, after 12 weeks, CB2-deficient lesions displayed increased lesional macrophage and smooth muscle cell (SMC) content and a ~2-fold reduction in lesional apoptosis. CB2-deficienct lesions also displayed reduced collagen content and elevated elastin fiber fragmentation that was associated with elevated levels of the extracellular matrix degrading enzyme, matrix metalloproteinase 9 (MMP9). These results demonstrate that although CB2 signaling does not affect atherosclerotic lesion size it does modulate lesional apoptosis, cellularity and ECM composition. Ldlr-/- and CB2-deficient Ldlr-/- mice were also subjected to daily treatments with Win55,212-2, a synthetic cannabinoid, over the last 2 weeks of an 8 week atherogenic diet to identify CB2-dependent and CB2-independent effects of cannabinoid receptor stimulation on atherosclerosis. Win55,212-2 did not affect hypercholesterolemia, aortic root lesion area, lesional macrophage infiltration, or ECM composition in either genotype but did significantly reduce total plasma triglyceride levels and lesional SMC content, independent of CB2. Surprisingly, lesional apoptosis was dose-dependently repressed by Win55,212-2 in Ldlr-/- mice by a CB2-dependent mechanism. All together, these results support the suggestion that CB2 may be a target for novel therapies aimed at modulating lesional apoptosis and cellularity to increase lesion stability and reduce the vulnerability to rupture.

Non-Small Cell Lung Cancer (NSCLC), which accounts for 80% of all lung cancer cases, is associated with resistance to chemotherapy and poor prognosis. Exploitation of NSCLC-upregulated pathways that can either be targeted by novel therapeutics or used to improve the tumour-delivery of current chemotherapeutics are required. Among the matrix metalloproteinases (MMPs) that are essential for tumour development, MMP10 is a potential candidate as a therapeutic target based on its expression and contribution to NSCLC development. This research aims to explore the expression and functions of MMP10 in the tumour microenvironment of NSCLC and evaluate the potential of MMP10 as a target for therapeutic intervention. Herein, MMP10 expression at gene and protein levels were analysed in a panel of NSCLC cell lines using RT-PCR and Western blotting analysis. To determine MMP10 functional relevance, an in vitro angiogenesis assay using cell conditioned media was carried out. To identify specific peptide sequences for the design of prodrugs rationalised to be MMP10 activated, in vitro substrate cleavage studies were performed using a mass spectrometry approach to differentiate between MMP10 and the structurally similar MMP3. This study demonstrates that MMP10 is highly expressed in NSCLC and that high levels of MMP10 are associated with induction of angiogenesis, a crucial process supporting tumour growth. In addition to the achievement of having been able to differentiate between closely similar MMP3 and MMP10 through carefully monitoring the hydrolysis rate of compound 444259 (a known MMP substrate), data generated herein provides the basis for further studies to exploit MMP10 as a prodrug-activator. / Full text was made available at the end of the embargo period, 12th Dec 2019

The deposition of β-amyloid peptides and of α-synuclein proteins is a neuropathological hallmark in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) subjects, respectively. However, there is accumulative evidence that both proteins are not exclusive for their clinical entity but instead co-exist and interact with each other. Here, we investigated the presence of a newly identified, pyroglutamate79-modified α-synuclein variant (pGlu79-aSyn)—along with the enzyme matrix metalloproteinase-3 (MMP-3) and glutaminyl cyclase (QC) implicated in its formation—in AD and in the transgenic Tg2576 AD mouse model. In the human brain, pGlu79-aSyn was detected in cortical pyramidal neurons, with more distinct labeling in AD compared to control brain tissue. Using immunohistochemical double and triple labelings and confocal laser scanning microscopy, we demonstrate an association of pGlu79-aSyn, MMP-3 and QC with β-amyloid plaques. In addition, pGlu79-aSyn and QC were present in amyloid plaque-associated reactive astrocytes that were also immunoreactive for the chaperone heat shock protein 27 (HSP27). Our data are consistent for the transgenic mouse model and the human clinical condition. We conclude that pGlu79-aSyn can be generated extracellularly or within reactive astrocytes, accumulates in proximity to β-amyloid plaques and induces an astrocytic protein unfolding mechanism involving HSP27.

Previously, we described prostate cancer (PCa) detection (83% sensitivity; 67% specificity) in seminal plasma by CE-MS/MS. Moreover, advanced disease was distinguished from organ-confined tumors with 80% sensitivity and 82% specificity. The discovered biomarkers were naturally occurring fragments of larger seminal proteins, predominantly semenogelin 1 and 2, representing endpoints of the ejaculate liquefaction. Here we identified proteases putatively involved in PCa specific protein cleavage, and examined gene expression and tissue protein levels, jointly with cell localization in normal prostate (nP), benign prostate hyperplasia (BPH), seminal vesicles and PCa using qPCR, Western blotting and confocal laser scanning microscopy. We found differential gene expression of chymase (CMA1), matrix metalloproteinases (MMP3, MMP7), and upregulation of MMP14 and tissue inhibitors (TIMP1 and TIMP2) in BPH. In contrast tissue protein levels of MMP14 were downregulated in PCa. MMP3/TIMP1 and MMP7/TIMP1 ratios were decreased in BPH. In seminal vesicles, we found low-level expression of most proteases and, interestingly, we also detected TIMP1 and low levels of TIMP2. We conclude that MMP3 and MMP7 activity is different in PCa compared to BPH due to fine regulation by their inhibitor TIMP1. Our findings support the concept of seminal plasma biomarkers as non-invasive tool for PCa detection and risk stratification.

Cardiovascular disease (CVD) is ranked the second leading cause of death in Canada, with 53,704 heart disease-related deaths documented in 2020 alone. After a patient sustains cardiac injury, such as a myocardial infarction (MI), the heart is often unable to undergo sufficient self-recovery for healthy cardiac regeneration and repair; this is largely attributed to fibrotic tissue development at the injury site and subsequent pathological ventricular remodeling. The prevalence of MI events has created a considerable demand to develop novel strategies for effective and safe post-MI therapies. Research has indicated that post-MI modifications interfere with endogenous cardiac repair mechanisms, resulting in a pathological state. After an infarction, there is an accumulation of methylglyoxal (MG) at the site of injury. It has been suggested that MG contributes to ventricular fibrotic development, however its underlying mechanism remains unclear. Additionally, the effects that the post-MI cardiac environment, specifically MG accumulation, has on post-MI therapies and biomaterials has not been sufficiently established. Accordingly, the primary focus of this research project is to elucidate the effects of MG on the collagen-rich extracellular matrix (ECM) of the heart and key cardiac cells involved in the repair process. Further, the interaction between MG and a promising collagen-based hydrogel therapy is investigated, exploring the effects of MG on the hydrogel’s degradative process. It was found that the MG modification of hydrogels did not alter the degradation rate. Additionally, the degradation products of hydrogels, and MG-modified substrates did not affect the properties and formation of myofibroblasts.

No description available.

No description available.

<p><strong> </strong>Fibrotic cataracts such as anterior subcapsular cataract (ASC) are induced by transforming growth factor beta (TGFβ). The mechanism which governs TGFβ-mediated ASC has not been elucidated. What is known is that TGFβ initiates the conversion of lens epithelial cells (LECs) to myofibroblast-like cells, through a process known as epithelial to mesenchymal transition (EMT). TGFβ-induced EMT leading to ASC has been associated with the upregulation of two matrix metalloproteinases (MMPs), MMP2 and MMP9. However, roles for either of these MMPs have yet to be established in ASC. To determine the involvement of MMP2 and MMP9 I used synthetic inhibitors in conjunction with an established <em>ex vivo </em>rat lens model initiated by TGFβ. The results demonstrated that co-culturing rat lenses with TGFβ and the matrix metalloproteinase inhibitor (MMPI), GM6001 or an MMPI specific for MMP2/9 suppressed ASC. Additionally, studies conducted on the conditioned media from these treatments revealed that TGFβ induces the cleavage of E-cadherin ectodomain which is suppressed by coculturing with either MMPI. To further delineate a role for MMP9 <em>in vivo</em>, ASC formation was examined in two models of lens specific TGFβ overexpression in the absence of functional MMP9. Adenoviral delivery of TGFβ to the anterior chamber of the eye in the absence of functional MMP9 resulted in complete suppression of ASC. Similarly, lens specific TGFβ overexpression in the absence of MMP9 suppressed ASC in 75% of mouse lenses. Additional studies determined that connective tissue growth factor is able to mediate ASC, albeit to a lesser degree than TGFβ.</p> / Doctor of Philosophy (PhD)

Yes / Despite significant preclinical promise as anticancer agents, vascular-disrupting agents have yet to fulfil their clinical potential due to systemic toxicities. ICT2588 is a tumour-selective MT1-MMP-targeted prodrug of azademethylcolchicine, ICT2552. We investigate activation of ICT2588 and subsequent release of ICT2552 in tumour cells, and examine its ability to induce G2/M cell cycle arrest. We also explore synergism between ICT2588 and ATR inhibition, since colchicine, in addition to its vascular-disrupting properties, is known to induce G2/M arrest, DNA damage, and trigger apoptosis. Several ATR inhibitors are currently undergoing clinical evaluation. The cellular activation of ICT2588 was observed to correlate with MT1-MMP expression, with selective release of ICT2552 not compromised by cellular uptake and prodrug activation mechanisms. ICT2588 induced G2/M arrest, and triggered apoptosis in MT1-MMP-expressing cells, but not in cells lacking MT1-MMP expression, while ICT2552 itself induced G2/M arrest and triggered apoptosis in both cell lines. Interestingly, we uncovered that the intracellular release and accumulation dynamics of ICT2552 subsequent to prodrug activation provided synergism with an ATR inhibitor in a way not observed with direct administration of ICT2552. These findings have important potential implications for clinical combinations of ICT2588 and DNA repair inhibitors.