Protein and gene expression analyses in bone marrow stem cells mediated restoration of myocardium after ischemic insult

PhDMyocardial Infarction (MI) is caused by occlusion of the coronary artery following atheromatous plaque rupture, the subsequent ischemia in the myocardium leads to myocyte necrosis unless treated quickly. Bone marrow derived stem cell treatment is a promising therapy for improving the outcome of patients with MI. The aim of this thesis was to study myocardial protein and gene expression changes in a rat ischemia/reperfusion (I/R) model in order to look for potential repair mechanisms of the myocardium triggered by endogenous bone marrow mononuclear cells (BMMNCs). Rat myocardial samples were obtained from three experimental groups: one group had a sham operation, the other two groups had undergone myocardial I/R injury induced by left anterior descending (LAD) coronary artery ligation followed by treatment with either a BMMNC preparation or PBS. Comparative proteomic analyses were carried out using 2D electrophoresis; differentially expressed proteins were identified using LC-MS/MS. Western blotting was used to confirm the most significant findings including expression of 14-3-3 epsilon protein. Global comparative gene expression profiling was performed using Illumina RatRef12 BeadChips and QPCR was used to validate the top results. Bioinformatic tools were used to assess the biology of the differentially expressed genes and proteins. Thirty-seven proteins were found to be differentially expressed in I/R injury compared to sham. These were primarily sarcomeric, energy production or stress response proteins and most were down-regulated. Expression levels were ‘corrected’ by BMMNC treatment for many of these proteins. Over 1500 genes were affected by I/R injury, 20 were affected by BMMNC treatment, and many of these were related to inflammation and apoptosis signalling and responses. The 14-3-3 epsilon protein was chosen for follow-up work as it presented as a good candidate for mechanistic involvement. This protein has many roles including interactions with the proapoptotic BCL2-associated agonist of cell death (Bad) protein. Western blotting was used to look at Bad expression and found it to be significantly increased in the Page 3 treatment group, although I could not reliably measure the expression of phosphorylated (serine 136) form of Bad. A preliminary pull-down assay was performed to look for binding partners of 14-3-3 epsilon. Two ATP synthase subunits, one of which is known to bind 14-3-3 epsilon, a protein involved in fatty acid β-oxidation and a protein of unknown function were found to bind. Further work will be required to follow up these findings and ascertain the exact role of 14-3- 3 epsilon in cardioprotection. In summary, my data supports the power of profiling methods to derive new candidates for a role in repair mechanisms in this therapeutic model

Protein discovery platform using deer antler as a model of mammalian regeneration

The ability to activate and regulate stem cells during wound healing and tissue regeneration is a promising field which could bring innovative approaches into regenerative medicine. The regenerative capacity of invertebrates has been well documented, however in mammals, stem cells that drive organ regeneration are rare. Deer antler is the only known mammalian structure that can annually regenerate to produce a complex tissue. The neural crest derived stem cells that drive this process result in antler growing at up to 2 cm/day. Deer antler appears to outweigh lower-order animal models when investigating the regulation of stem cell-based regeneration. Pleiotrophin (PTN) is a multifunctional heparin-binding growth factor; the PTN gene is found highly expressed within the active antler stem cell tissues. The studies presented in this thesis aimed to examine the location of stem cells during antler growth, the proteomic profiles of different pools of stem cells involved in antler generation and regeneration, and the role of the PTN growth factor family in stem cell regulation. The differentially expressed proteins between cells derived from stem cell niches involved in antlerogenesis (antlerogenic periosteum) and regeneration (potentiated and dormant pedicle periosteum), and cells derived from deer facial periosteum as a control (n = 3) were identified using 2D-DIGE-based quantitative proteomics. Label-free mass spectrometry was further used to detect the protein expression profiles of antler stem cell tissues under different stages of activation and included: dormant pedicle periosteum, growth centre, post-active stem cells from mid-beam periosteum, and control facial periosteum (n = 3). Mesenchymal stem cell markers CD73, CD90 and CD105, along with PTN/midkine (MDK) growth factors and their receptors (PTPRZ, ALK, NOTCH2, ITGAV and ITGB3), were examined in antler tissues using immunohistochemistry. In vitro effects of PTN on proliferation and osteogenic differentiation of antler stem cells were also investigated. Ninety-two differentially expressed proteins were identified by 2D-DIGE. Bioinformatic analysis indicated the epithelial-mesenchymal transition process may participate in the initiation of wound healing and subsequent antler regeneration; cell mobility was highly involved during antler regeneration; energy and nucleotide metabolism may however be less active in antler regeneration as compared to that in antler generation phase. Immunohistochemistry confirmed the central role of stem cells in the development of this mammalian structure by localising the mesenchymal stem cell markers within the antler growth centre. Label-free quantification distinguished unique markers of dormant (6), active (87) and post-active (3) antler stem cells showing that the greatest number of proteins was exclusively found in the active stem cell tissue. There were only 12 proteins detected with expression levels that significantly differed between tissue with dormant stem cells and the control tissues. Protein profiles of these two groups showed that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. One hundred fifty-three significantly regulated proteins were found between antler stem cell tissues under different activation stages; activation of antler stem cells was associated with up-regulation of a number of canonical pathways and molecular/cellular functions such as Hippo and canonical Wnt signalling. PTN was identified as the dominant growth factor in the PTN/MDK family with higher expression levels in the antler growth centre. High expression of PTPRZ and ALK co-localised with PTN suggested their potential interactions. The high levels of PTN and PTPRZ also reflected the antler stem cell activation status during the regenerative process. When antler stem cells were cultured in vitro under the normoxic condition, no PTN was expressed and exogenous PTN did not induce differentiation or proliferation but rather stem cell maintenance. In summary, this research project explores potential biomarkers for mammalian stem cells, as well as the key proteins, biological processes and pathways involved in stem cell maintenance, development and activation during antler generation and regeneration

Italian Proteomics Association, 5th Annual National Conference Abstract Volume

Abstract volume of the Italian Proteomics Association 5th Annual national conferenc

Testing the inside-out theory of multiple sclerosis aetiology using the cuprizone-induced demyelination model

Multiple sclerosis (MS) is a common disabling neurological disorder affecting about 2.5 million people worldwide. There are two competing theoretical aetiologies of the disease: First, an ‘outside-in’ theory in which peripheral factors (autoreactive T-cells) traverse the blood brain barrier (BBB) leading to central nervous system (CNS) demyelination. Second, an ‘inside-out’ theory in which central demyelination causes BBB disruption leading to autoimmune response. Cuprizone (CPZ)-feeding in mice induces oligodendrocyte loss (oligodendrocytosis), demyelination and reactive gliosis of astrocytes and microglia in the central nervous system (CNS), similar to that seen in MS; however, the CPZ-induced pathology lacks T-cell involvement in the CNS, a hallmark of MS, in particular by CD8+ T-cells. This thesis explored whether incremental modification of the standard CPZ mouse model would yield a pattern of disease development and progression more closely resembling that seen in human MS. The first modification tested the hypothesis that blood brain barrier (BBB) disruption (using pertussis toxin, PT) would facilitate a T-cell-mediated immune response to the myelin antigens in the CNS of CPZ-fed mice. This was tested by using low (0.1%) and standard (0.2%) CPZ-feeding for 5 weeks (Study I) and also assessing whether similar effects can be initiated by inducing a slow progressive demyelination by prolonged feeding of 0.1% CPZ for 12 weeks (Study II). The use of 0.1% CPZ yields a comparable CNS pathology to 0.2% but less effect on splenic T-cell (CD4 and CD8, CD4/8) levels and no change in spleen mass; thus, only 0.1% CPZ was used in Study II. Although oligodendrocytosis, demyelination and gliosis were evident in Studies I and II, no T-cells were detected in the CNS. Proteomic analysis of whole brain tissue reveals that CPZ has changed the abundance of many proteoforms that are directly involved in metabolism and suppression of T-cell function. Importantly, 0.1% and 0.2% CPZ-feeding lead to a reduction in splenic T-cells, indicating that the peripheral immune system is suppressed due to the effects of CPZ-feeding. In study III, castration (Cx, orchiectomy) is used to protect the peripheral immune system against the dose dependent (0.2%>0.1%) effects of CPZ-feeding on thymus and spleen size (and T-cell levels) yet produces the same amount of demyelination and gliosis in the MCC. Study IV demonstrates that combining Cx induced preservation of the thymus and spleen with 0.1% CPZ-feeding and blood brain barrier disruption result in CD8+ T-cell recruitment into the brain and spinal cord. In study V, 0.1% CPZ-feeding in gonadally intact (Gi) female mice induces peripheral immune organ atrophy, T-cell signal suppression, demyelination and gliosis indistinguishable from that seen in males, but no CD8+ T-cell infiltration into the CNS. Taken together, suppression of the peripheral immune system in male and female mice by CPZ-feeding explains why CPZ-induced demyelination does not result in the recruitment of peripheral immune cells into the CNS. Moreover, these findings indicate that a short period (2 weeks) of 0.1% CPZ-feeding in Cx males (where the peripheral immune structures are preserved), induces both demyelination and glial activation that are sufficient to activate and recruit CD8+ cells to the CNS, in effect initiating an ‘inside-out’ T-cell mediated immune response. The CD8+ T-cell recruitment into the CNS of CPZ-fed mice is a new variant of the CPZ model that should prove useful to explore the earliest events involved in CNS demyelinating diseases like MS

Proteomic identification of putative biomarkers of radiotherapy resistance

BackgroundCurrently, tumour response to radiotherapy cannot be predicted meaning that those patients with tumours resistant to the therapy endure the harmful side effects associated with ionising radiation in the absence of therapeutic gain. The aim of this project was to identify protein biomarkers predictive of radiotherapy response using comparative proteomic platforms to study radioresistant cell line models. The identification of such biomarkers will enable radiotherapy to be tailored on an individual patient basis and hence increase treatment efficacy.MethodsSeven radioresistant (RR) cell line models derived from breast, head and neck (oral), and rectal cancers were investigated to identify differentially expressed proteins (DEPs) associated with radiotherapy resistance. This included the establishment of 2 RR rectal cancer cell line models and the proteomic analysis of 2 RR oral cancer cell lines and 2 RR rectal cancer cell lines. Proteomic analysis included 3 different platforms, namely antibody microarray, 2D MS and iTRAQ. Data mining of all biomarker discovery data, from all 7 novel RR cell lines was carried out using Ingenuity Pathway Analysis (IPA) which identified canonical pathways associated with the data. Protein candidates from selected canonical pathways were confirmed by western blotting and assessed clinically using immunohistochemistry.ResultsFollowing the combination of all biomarker discovery data for all 7 RR cell lines, 373 unique DEPs were successfully mapped onto the Ingenuity Knowledge Base, generating 339 canonical pathways. Of these, 13 of the most relevant pathways were selected for further interpretation. Several proteasomal subunits were identified during the biomarker discovery phase and were mapped onto the protein ubiquitination pathway by IPA. DR4, was identified in 4/7 RR cell lines and was mapped onto the death receptor signalling pathway by IPA. Radiotherapy is typically thought to induce cellular apoptosis via the intrinsic (mitochondrial) pathway, therefore the repeated identification of the DR4 protein involved in the extrinsic apoptotic pathway has potentially lead to the discovery of a novel relationship between radiotherapy and the extrinsic death receptor pathway. The differential expression of both the 26S Proteasome and DR4 were confirmed by western blotting. Clinical assessment using immunohistochemistry revealed a significant association between expression of the 26S Proteasome and radioresistance in breast cancer.DiscussionA large number of DEPs which may be associated with radiotherapy resistance in breast, oral and rectal cancers have been identified using comparative proteomic platforms. The protein ubiquitination pathway and the death receptor signalling pathway may play a significant role in radioresistance and proteins within these pathways may be putative biomarkers of radiotherapy response

Discovery and application of colorectal cancer protein markers for disease stratification

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise methods of CRC stratification are needed. The intracellular protein expression from 28 CRC primary tumours and corresponding normal intestinal mucosa was analysed using saturation-DIGE/MS and Explorer antibody microarrays. Changes in protein abundance were identified at each stage of CRC. Proteins associated with proliferation, glycolysis, reduced adhesion, endoplasmic reticulum stress, angiogenesis, and response to hypoxia represent changes to CRC and its microenvironment during development. Molecular changes in CRC cells and their microenvironment can be incorporated into clinic-pathological data to help sub-classify tumours and personalise treatment. DotScan antibody microarray analysis was used to profile the surface proteome of cells derived from 50 CRC samples and corresponding normal intestinal mucosa. Fluorescence multiplexing enabled the analysis of two different sub-populations of cells from each sample: EpCAM+ cells (CRC cells or normal epithelial cells in normal mucosa) and CD3+ T-cells (tumour-infiltrating lymphocytes). Unsupervised hierarchical clustering of the CRC and T-cell surface profiles defined four clinically relevant clusters, which showed some correlation with histopathological and clinical characteristics such as cancer cell differentiation, peri-tumoural inflammation and stimulation of infiltrating T-cells. The observed relationship between the surface antigen expression profiles of patients’ CRC cells and their corresponding tumour infiltrating T-cells suggests that CRC surface proteins may play a direct role in influencing the activity (and hence surface protein expression) of neighbouring T-cells and/or vice versa. We conclude that the application of surface profiling may provide improved patient stratification, allowing more reliable prediction of disease progression and patient outcome

Discovery and application of colorectal cancer protein markers for disease stratification

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise methods of CRC stratification are needed. The intracellular protein expression from 28 CRC primary tumours and corresponding normal intestinal mucosa was analysed using saturation-DIGE/MS and Explorer antibody microarrays. Changes in protein abundance were identified at each stage of CRC. Proteins associated with proliferation, glycolysis, reduced adhesion, endoplasmic reticulum stress, angiogenesis, and response to hypoxia represent changes to CRC and its microenvironment during development. Molecular changes in CRC cells and their microenvironment can be incorporated into clinic-pathological data to help sub-classify tumours and personalise treatment. DotScan antibody microarray analysis was used to profile the surface proteome of cells derived from 50 CRC samples and corresponding normal intestinal mucosa. Fluorescence multiplexing enabled the analysis of two different sub-populations of cells from each sample: EpCAM+ cells (CRC cells or normal epithelial cells in normal mucosa) and CD3+ T-cells (tumour-infiltrating lymphocytes). Unsupervised hierarchical clustering of the CRC and T-cell surface profiles defined four clinically relevant clusters, which showed some correlation with histopathological and clinical characteristics such as cancer cell differentiation, peri-tumoural inflammation and stimulation of infiltrating T-cells. The observed relationship between the surface antigen expression profiles of patients’ CRC cells and their corresponding tumour infiltrating T-cells suggests that CRC surface proteins may play a direct role in influencing the activity (and hence surface protein expression) of neighbouring T-cells and/or vice versa. We conclude that the application of surface profiling may provide improved patient stratification, allowing more reliable prediction of disease progression and patient outcome

Risk stratification in atherosclerotic cartoid stenosis

Introduction: Key trials and a Cochrane systematic review in asymptomatic carotid stenosis have highlighted the need to identify a high-risk subgroup of patients with carotid stenosis who may benefit from intervention. Traditionally, this risk stratification has considered structural imaging and clinical factors. However, using only these approaches, still a significant number of patients are missed. Biological attributes are acknowledged as key determinants of thrombo-embolic events. Functional and hybrid structural-functional imaging, and circulating biomarkers allow exploration of plaque biology non-invasively, in vivo. The importance of innate immunity in atherosclerosis is now established, with a recent interest in macrophage phenotypic polarisation in atherosclerosis supported by in vitro and experimental data, with the hypothesis of an M1 macrophage predominance associated with unstable plaques. The emergence of systems biology has been seen to facilitate understanding of biological pathways and generate hypotheses, although the utility of this approach for the examination of human atherosclerosis tissue has not been fully explored. Aims: (i) To employ functional imaging to probe carotid atherosclerosis in vivo; (ii) to assess the plaque microenvironment in determination of the balance of macrophage populations in unstable compared with stable atherosclerosis; (iii) to investigate whether late phase (LP-) contrast enhanced ultrasound (CEUS) reflects plaque biological features; (iv) to examine the utility of systems biology techniques in distinguishing symptomatic from asymptomatic carotid atherosclerosis tissue, and in hypothesis generation; and (v) to evaluate a putative biomarker for carotid atherosclerosis and plaque vulnerability. Methods: Patients with carotid stenosis, both symptomatic and asymptomatic, have undergone systematic collection of data, fresh carotid endarterectomy (CEA) specimens, and plasma. Thirty-two patients with 36 carotid stenoses underwent 11C-PK11195 PET/CT. Thirty-seven patients had dynamic (D-) and LP-CEUS carotid imaging. CEA specimens were assessed by immunohistochemical techniques, as well as atheroma cell culture with supernatant multi-analyte profiling (MAP). MAP data was subject to Ingenuity Pathway Analysis. CEA specimens were further examined using systems biology methodologies: transcriptomics with Affymetrix Human Exon 1.0 ST arrays; proteomics and lipidomics by liquid chromatography (LC) coupled to tandem triple quadrupole mass spectrometry (MS); and metabolite profiling by nuclear magnetic resonance and LC-MS. Furthermore, venous and arterial plasma was quantified for the lysozyme, a putative biomarker in carotid atherosclerosis. Results: 11C-PK11195 PET allowed the non-invasive quantification of intraplaque inflammation in patients with carotid stenoses and, when combined with CTA, provided an integrated assessment of plaque structure, composition and biological activity. 11C-PK11195 PET/CT distinguished between recently symptomatic vulnerable plaques and asymptomatic plaques with a high positive predictive value. D-CEUS and LP-CEUS (at a cut-off of zero) was able to distinguish symptomatic and asymptomatic plaques. Atheroma cell culture and supernatant MAP revealed that symptomatic human atherosclerotic carotid disease is associated with a cytokine and chemokine pattern consistent with the predominance of pro-inflammatory M1-type macrophage polarisation. Furthermore, IFNγ signatures are observed, including the novel finding of CCL20 with its significant elevation in symptomatic atherosclerosis. MAP of supernatants from patients who had undergone ipsilateral carotid LP-CEUS revealed significantly higher levels of IL6, MMP1 and MMP3, as well as greater CD68 and CD31 immunopositivity, in those with high (≥0) compared with low (<0) LP-CEUS signal. This suggests that LP-CEUS was able to reflect plaque biology. Transcriptomic analysis was able to clearly separate stenosing plaque and intimal thickening, as well as unstable and stable atherosclerosis, finding differential expression and alternative splicing of interferon regulatory factor 5 between stenosing plaque and intimal thickening. Proteomic analysis of the salt extract fraction from carotid atherosclerotic plaques identified 2,470 proteins implicated in 33 bio-molecular functions and having their origins previously described in 14 different cellular compartments. There were 159 proteins which, based upon the number of assigned spectra, were significantly different between symptomatic and asymptomatic atherosclerosis. Through lipidomic analysis, 150 lipid species from 9 different classes were identified, of which 24 were exclusive to atherosclerotic plaques. A comparison of 28 carotid endarterectomy specimens revealed differential lipid signatures of symptomatic compared with asymptomatic lesions, as well as stable and unstable plaque areas. Similarly, LC-MS metabolite profiling of organic plaque extract was able to separate symptomatic from asymptomatic atherosclerosis. Arterial and venous plasma lysozyme levels were seen to distinguish individuals with carotid atherosclerosis from matched control subjects. Furthermore, arterial plasma lysozyme levels were significantly higher in patients with symptomatic than asymptomatic carotid stenosis. Conclusions: These findings support the use of hybrid structural-functional imaging, and the utility and use of a systems biology approach in identifying significantly different and biologically relevant variations in atherosclerosis tissue, and in hypothesis generation for further study. The data presented concurs with recent reports in the literature linking the lipidic/organic component of atherosclerosis with the generation of a pro-inflammatory plaque microenvironment prone to lesion development, instability and the complications thereof. The importance of innate immunity has been highlighted with the demonstration of a predominance of M1 macrophage polarisation and evidence of Th17/IL17 signalling in unstable atherosclerosis. It is hoped that this work will contribute to the ongoing refinement of multi-factorial risk stratification in carotid atherosclerosis