Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted. Includes bibliographical references.

A Research Report submitted to the Faculty of Medicine, University of the Witwatersrand, in part fulfilment of the requirements for the degree of Master of Medicine in the branch of Haematology Johannesburg, October 1997 / Venous thromboembolic disease is a common health problem. It contributes considerably to morbidity as well as to mortality. Thrombosis usually occurs due to an underlying risk factor which may be environmental or genetic in origin. The recently described activated Protein C (APC) resistance is the commonest cause of familial thrombophilia documented to date. The molecular lesion is a single point mutation in the factor V (FV) gene which abolishes a cleavage site whereby it is normally inactivated by APC. This defect, termed the FV Leiden mutation, is highly prevalent in normal Caucasian populations. Although it would appear to have arisen due to a founder effect, there is a paucity of data concerning non-Caucasian populations. / IT2018

Le choc septique est la principale cause de mortalité dans les services de réanimation. Les glucocorticoïdes (GC) et la protéine C activée (APC) sont deux traitements adjuvants recommandés au cours du choc septique. Ce travail a pour objectif d'évaluer l'impact de la combinaison d'APC et des GC sur les paramètres hémodynamiques et la survie. Le sepsis expérimental se caractérise par une hypotension artérielle avec acidose lactique et une hyporéactivité vasculaire. L'administration de Dexa et/ou d'APC permet de diminuer les taux de lactates, d'interleukines et de nitrate/nitrite. Chez les groupes traités, la contraction est améliorée ainsi que la relaxation vasculaire des aortes et des artères mésentériques. L'administration d'APC et de Dexa, seul ou en association, entraine une diminution de l'expression induite d'iNOS et la restauration de la voie Akt. La combinaison APC et Dexa améliore le temps de survie de façon synergique. Nos résultats suggèrent que l'APC et les GC devraient être réévalués en association dans le traitement du choc septique / Sepsis remains the major cause of death in intensive care units. International guidelines for management of severe sepsis and septic shock recommend both stress-dose steroid therapy and recombinant activated protein C (APC). The aims of the present study were to compare the effects of APC and dexamethasone (Dexa) alone as well as in combination in resuscitated septic shock on survival, hemodynamics, and vascular reactivity. Sepsis was associated with a decrease in mean arterial pressure, elevation in plasma lactate and nitrite/nitrate concentration. Administration of APC, Dexa, and their combination improve arterial pressure and decrease lactate and nitrite/nitrate concentration. Both APC and Dexa improved arterial contractility and endothelial dysfunction resulting from septic shock in rats. The expression of iNOS was significantly reduced by the administration of Dexa, APC, and combination therapy. All treatments restore the Akt pathway. Moreover, their combination increased the length of survival. These findings suggest that APC and glucocorticoids should be further re-evaluated in combination in septic shock

Severe Acute Pancreatitis (SAP) is the rapid onset of inflammation within the pancreatic organ. Unlike the milder form of this illness, SAP is associated with a high mortality and morbidity. No significant reduction in the outcomes of this disease has been made since the implementation of organ supportive management over two decades ago. This is due to difficulties in distinguishing between the milder form of the disease in the early period of the onset of symptoms when clinical intervention is most likely to prevent complications and death. Clinical equipoise exists in the management of one of these complications, namely Abdominal Compartment Syndrome (ACS) as the conventional management of surgery runs contrary to published evidence showing early abdominal surgery deteriorates clinical outcomes. Aims: Validation of the potential use of the Early Warning Score (EWS) as a predictor of SAP. Evaluation of the evidence for recombinant human protein C (Xigris™) in the early treatment of SAP. Determination of the safety profile of Xigris™ when given early in SAP. To determine if surgical management of ACS in SAP is of significant benefit compared to conventional management alone. Methods: Four studies were performed: A prospective observational study assessing the median EWS of patients admitted with acute pancreatitis; a systematic review of published evidence reporting the use of Xigris™ in SAP; a prospective cohort study using a 24 hour infusion of Xigris™ early in patients diagnosed with SAP and a pilot randomized controlled trial of targeted decompression in patients with ACS complicating SAP. Results: The highest EWS values for 130 patients with acute pancreatitis within the first 3 days of admission were not shown to have significant sensitivity and specificity in predicting an unfavourable outcome. A review of the published literature between from January 1985 to January 2011 supported the further investigation of Xigris™ as a treatment for SAP. No significant adverse events or differences in outcomes were evident in 19 patients who received a 24-hour infusion of Xigris™ early in SAP compared to matched historical controls. 22 patients were screened for the development of ACS. No patient developed ACS and consequently no randomization to either treatment arm was possible. Conclusion: With the recent advent of an updated classification system for the severity of acute pancreatitis, further prospective evaluation of the use of EWS in clinical practice is warranted. The results of the Phase 1 clinical trial of Xigris™ didnot reveal significant safety issues that might preclude the further investigation of Xigris™ as a specific therapy early in the onset of SAP. The absence of ACS inpatients with SAP lends support to a theory that ACS may be an epiphenomenon in the course of SAP.

Sepsis has been defined as a systemic response to an infection. With an incidence of 3 per 1000 population per year or about 750 000 cases a year, this syndrome ranks as the 10th leading cause of death in the United States (1). Increasing severity of sepsis correlates with increasing mortality, which rises from 30-40% for severe sepsis up to 40-60% for septic shock. This thesis examines the effectiveness of adjunctive therapies, including activated protein C, hypercapnia and acidosis, and sodium selenite, in a clinically relevant ovine model of septic shock. The results from these studies can provide valuable information for future clinical trials on sepsis.<p>This thesis is divided into four sections: 1) sepsis overview; 2) an autologous fecal peritonitis model in sheep and its evaluation; 3) the series of studies on adjunctive therapeutics; and 4) ongoing studies and future perspective.<p>In the first section, a broad overview gives a rough introduction to delineate many aspects of sepsis syndrome such as terminology, etiology, epidemiology, pathophysiology and current guidelines for management. Hemodynamics in sepsis are especially elaborated since these are major observations throughout the studies presented later.<p>In the second section, the general characteristics of the sepsis models used in this thesis are elucidated. Data on hemodynamics, lung mechanics, gas exchange, etc. are presented to feature the ovine peritonitis model. The results of laboratory examinations for hematology, coagulation, bacteriology, biochemistry and hormonology are also presented. And then, I review currently used sepsis models with regards to their advantages and disadvantages.<p>The third section discusses three studies with their objectives, the methods used, the major findings, and the potential clinical implications.<p>9<p>1) Beneficial effects of recombinant human activated protein C in experimental septic shock. Activated protein C has a multitude of beneficial effects in severe sepsis and septic shock, including anti-inflammation, anti-coagulation, profibrinolysis, anti-apoptosis and endothelial protection. A clinical Phase III trial demonstrated that the administration of recombinant human activated protein C improved survival in patients with severe sepsis. However, doubts on the protective effects of activated protein C have persisted and been refueled by the recently published negative trials in less severely ill patients and in children. In the light of these ambiguities and uncertainties, we reinvestigated the effects of activated protein C in experimental septic shock.<p>2) Acute hypercapnia improves indices of tissue oxygenation more than dobutamine in septic shock. Hepercapnia has been found to possess beneficial effects in diverse acute inflammatory states independent of protective lung mechanics. To prove the hypothesis that acute hypercapnia has similar or superior hemodynamic effects to those of a dobutamine infusion, which may be particularly relevant in the presence of hemodynamic instability associated with respiratory failure, we investigated the effects of hypercapnia, which induced by inspiring extrinsic carbon dioxide in experimental septic shock.<p>3) High bolus dose of sodium selenite prolongs survival in an ovine model of septic shock. Selenite has both pro- and anti-oxidant effects. The administration of high dose sodium selenite may improve survival in septic shock patients. The benefit may be greater with the administration of a bolus (to achieve higher concentrations) rather than a continuous infusion. To test this hypothesis, we examined the effects of a high dose bolus administration of sodium selenite in experimental septic shock.<p>The fourth and final section talks about currently ongoing studies and offers some perspective on future direction. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Effective management of acute pancreatitis has for centuries eluded mankind. The disease has a wide spectrum of presentation; the milder form is usually a self limiting condition, whereas the severe form presents as a highly morbid and frequently lethal attack. The ability to predict disease progression on admission would aid in the comprehensive and multidisciplinary management of patients. The perfect predictor of disease progression has been an elusive factor hindering the management of the disease. On systematically reviewing literature and identifying appropriate biochemical markers in predicting progression of acute pancreatitis, the ideal predictor would be a combination of biochemical, clinical and contemporary organ dysfunction scoring systems. Early prediction of disease progression however, is important in the better management of the disease. The pathophysiological changes of acinar cell injury and death are the earliest events that occur in acute pancreatitis. Identification of potential pharmacological interventions offered through valuable insight in to experimental and clinical acute pancreatitis may lead on to the development of various natural and synthetic potential disease modifiers. Green Tea Extracts (GTE) consumed in many parts of the world has been examined as a potential therapeutic medication. Experimental results have demonstrated the effect of GTE on the oxidative pathway significantly ameliorating the effects of pancreatic injury. The various green tea catechins especially Epigallocatechin-3- gallate (EGCG) can perhaps be useful lead compounds for new drug discovery. With no specific targeted therapy for severe acute pancreatitis at present, various medications have been tested. The possibility of targeting initial acinar cell injury may not be a feasible option as patient presentation and management would usually be after this phase. As the disease progresses, severe acute pancreatitis is characterised by inflammation and necrosis. The hypothesis of preserving pancreatic parenchymal microvascular patency and thus ameliorating pancreatic injury through the early administration of recombinant human Activated Protein C (rhAPC) has identified a potential treatment for acute pancreatitis. rhAPC converted from its inactive precursor, protein C, by thrombin acts through fibrinolysis and inhibition of thrombosis. Studies on rhAPC in experimental acute pancreatitis examined the modulation of rhAPC on inflammatory markers, morphology, microvascular thrombosis and apoptosis. The encouraging results from initial experimental work helped set up the Phase 2 clinical trial of administering rhAPC early on in severe acute pancreatitis. Prior to taking this significant step from bench to bed side, the variation in functional protein C levels with the severity of the disease was examined as a precursor to the Phase 2 trial.

The current findings indicate that activated protein C (aPC) can induce FOXP3 generation and regulatory T cells (Tregs) through epigenetic modulation and metabolic reprogramming. The current findings show that preincubation of T cells with aPC increased the frequency of Treg markers, CD4+FOXP3+ T cell frequency and suppressive function of T-cells, suggesting an increase of Tregs. The emergence of FOXP3 expression and Treg-like characteristics is linked to alterations in the epigenetic profile of T cells, characterized by a decline in overall DNA methylation, a decrease in the repressive histone marks H3K27me3 , and a reduced methylation of the FOXP3 promoter region. In addition, the induction of Tregs by aPC is accompanied by changes in mitochondrial metabolism. T lymphocytes that were preincubated with aPC exhibit a decline in mitochondrial respiration, a decrease in mitochondrial membrane potential, and a transition towards metabolic quiescence. The metabolic alterations are associated with a reduction of crucial metabolites, specifically α-ketoglutarate (αKG) and glutamine, that are known to regulate T cell function and epigenetic regulation. Reversal of the aPC-induced Treg-like phenotype and associated altered mitochondrial metabolism can be achieved through the supplementation of exogenous αKG or glutamine, supporting a functional interaction of these reduced metabolites and altered T-cell function. The current results show that chronically elevated levels of aPC in transgenic APChigh mice lead to a higher incidence of CD4+FOXP3+ Tregs in the spleen without alterations in thymic Tregs (tTregs), thereby indicating that aPC facilitates the development of Tregs in peripheral lymphoid organs but does not influence primary T-cell development. T cells derived from APChigh mice display a decrease in mitochondrial metabolism, which is consistent with the findings observed in vitro. Supplementation of αKG in cultured T cells derived from APChigh mice restores mitochondrial function and decreases the frequency of CD4+FOXP3+ Tregs. These findings support a model in which aPC alters T-cell metabolism, possibly by reducing expression of glucose (GLUT1) and glutamine (ASCT2 and SNAT1) transporters and thus the availability of metabolic substrates in T-cells. The reduced availability of these substates alters epigentic gene-expression and favors Treg development. In summary, the findings indicate that activated protein C (aPC) promotes the induction of regulatory T cells (Tregs) via metabolic modulation and altered epigenetic gene-expression. The results provide new insights into the long-lasting effects of aPC and indicate the possibility of aPC as a therapeutic target for regulating immune responses and fostering immune tolerance in diverse pathologies. Additional research is necessary to investigate the potential therapeutic applications.

Despite medical advances in the last decades, diabetic kidney disease (DKD) remains a major therapeutic challenge. DKD, the major microvascular complication in diabetic patients, is the most common cause of chronic kidney disease (CKD) and end-stage kidney disease (ESKD) requiring dialysis worldwide. In recent years, new therapeutic approaches for the treatment of DKD in addition to stringent blood glucose control and inhibition of angiotensin-signaling have been established. These new therapeutic options include sodium glucose cotransporter-2 inhibitors (SGLT2i), GLP-1 agonists, and nonsteroidal mineralocorticoid receptor antagonists. Despite their promising positive outcomes regarding kidney function, their long-term effects through the course of the disease remain unknown. Additionally, recent data show that about 50% of patients with DKD do not respond to these new therapeutics even if given in combination and there is a lack of therapies that can reverse the already established DKD. The continuous progression of diabetic complications (including DKD) despite normalization of blood glucose levels is referred to as the hyperglycemic memory. This phenomenon represents an unsolved medical problem in the field of diabetes management and remains without specific treatment options. Several theories have been proposed to explain the persistence of hyperglycemia-induced cellular dysfunction despite blood glucose normalization, and epigenetic regulation of gene expression has been proposed as the key pathomechanism driving the hyperglycemic memory. In this study, we used a combination of animal models, analyanalysesuman tissue and biofluid samples, in addition to in vitro mechanistic studies to identify therapeutically targetable pathways for the hyperglycemic memory in the context of DKD. To experimentally address the hyperglycemic memory, we used a model of hyperglycemia reversal in type-1 (STZ) and type-2 (db/db) diabetic mice. Hyperglycemia was reversed using SGLT2i (STZ and db/db mice) or insulin (STZ mice). We started by characterizing the functional and histological changes associated with hyperglycemic memory including persistent albuminuria, tubular hypertrophy, and fibrosis. To identify the pathways associated with the hyperglycemic memory in both models, we conducted bulk RNA sequencing from the kidneys and identified a large number of genes that were persistently up- or downregulated despite blood glucose normalization and hence possibly contributing to the hyperglycemic memory. Among these genes, we identified the cyclin-dependent kinase inhibitor p21 (Cdkn1a), which is known to regulate cellular senescence, as a primary candidate associated with the hyperglycemic memory, where its expression remained persistently upregulated despite blood glucose lowering in both diabetes models. Further investigations confirmed the “memorized” expression of p21 across DKD animal models and in cell lines in vitro, pinpointing the tubular epithelium as the specific cell type where this phenomenon occurs. Using a multimarker approach, we identified a persistent tubular senescence phenotype associated with p21 induction regardless of the intervention to reduce blood glucose levels in murine DKD models. Subsequent analyses aimed to scrutinize the relevance of this finding in the context of human DKD. We found induction of tubular p21 expression and senescence in human DKD biopsies compared to controls or to diabetic patients without kidney dysfunction. Furthermore, p21 was readily detected in the urine of DKD patients in a large cross-sectional cohort whic,h was increased with the severity of the disease and was negatively correlated with kidney function. Interestingly, urinary p21 levels remained persistently elevated despite different interventions to reduce blood glucose levels (SGLT2i or fasting-mimicking diet), corroborating its utility as a biomarker for the hyperglycemic memory in DKD. Next, we further elucidated the sequence of eventeventshyperglycemia to the induction of p21 and subsequent kidney damage, demonstrating that elevated blood glucose decreases DNA methyltransferase 1 (DNMT1) expression, resulting in hypomethylation of the p21 promoter and increased p21 expression. The induction of p21 expression triggers a senescence phenotype, that contributes to tubular damage and fibrosis. Suppression of tubular DNMT1 expression was sufficient to induce p21 in tubular cells, corroborating the importance of this epigenetic mechanism for the glucose-induced persistence of p21 expression. In order to investigate possible translational implications, we studied the potential of reversing the hyperglycemic memory in DKD. We employed the cytoprotective protease activated protein C (aPC), a disease resolving mediator associated with DKD protection. aPC in conjunction with the blood glucose lowering drug SGLT2i induced the expression of DNMT1 leading to promoter remethylation and suppression of the persistent tubular p21 expression. Notably, SGLT2i alone had no effect on p21 expression. The combined action of SGLT2i and aPC effectively counteracted albuminuria, tubular damage, senescence, and fibrosis in a murine DKD model. The protective effects of aPC were confirmed by investigating TMPro/Pro mice, in which thrombomodulin-mediated protein C activation is hampered resulting in reduced aPC levels. Induction of persistent hyperglycemia in these mice elevated p21 expression in association with aggravated kidney damage compared to wild-type mice. Superimposed deficiency of p21 in the aforementioned aPC-deficient mice (TMPro/Pro x p21-/-) alleviated the tubular injury and senescence phenotype, providing experimental in vivo evidence for a regulation of the hyperglycemic memory in DKD by the interaction of p21 by aPC levels. To confirm that aPC regulates DNMT1 and hence p21 in an epigenetic manner, we targeted DNMT1 in aPC-treated mice using the pan DNMT inhibitor 5-aza-2'-deoxycytidine or a specific vivo morpholino against DNMT1. Both approaches abolished aPC’s protective effects in a murine DKD model and inhibited its ability to reduce p21 promoter hypomethylation and hence its expression. Exploiting the cytoprotective properties of aPC by using the mutant 3K3A-aPC, which lacks the anticoagulant ability, or the chemical compound parmodulin-2 that mimics the biased signaling of aPC via the G protein-coupled receptor PAR1, was enough to reduce the hyperglycemia-induced p21 expression and the associated tubular senescence in murine DKD. The findings of this study uncover an important role of p21 in exacerbating renal damage under diabetic conditions, suggesting that p21 not only serves as a marker of cellular senescence but actively contributes to the persistence of DKD by mediating the hyperglycemic memory. By addressing the root causes of hyperglycemic memory, such as the epigenetic modifications that perpetuate p21 expression, it may be possible to halt or even reverse the progression of DKD. The feasibility of this approach was demonstrated by exploiting cytoprotective aPC signaling, which restored DNMT1 expression and reduced p21 expression. Thus, targeting the hyperglycemic memory in DKD may be feasible in general and may be specifically achieved by targeting cytoprotective aPC signaling.:Table of contents Table of contents 2 List of figures 5 List of tables 6 List of abbreviations 7 1. Introduction 9 1.1 Diabetes mellitus 9 1.2 Diabetic complications 9 1.3 Diabetic kidney disease (DKD) 10 1.3.1 Clinical presentation and staging of DKD patients 10 1.3.2 Cellular dysfunction in DKD 12 1.4 The hyperglycemic memory: a new challenge in DM management 14 1.4.1 Hyperglycemic memory in DKD 14 1.4.2 Mechanisms of the hyperglycemic memory 18 1.5 Cellular senescence in DKD 20 1.5.1 Features of senescent cells in DKD 20 1.5.2 Tubular cell senescence in DKD 23 1.6 Therapeutic management of DKD 23 1.6.1 SGLT2 inhibitors 24 1.6.2 Other therapeutic options for DKD 25 1.7 Coagulation proteases and their receptors in DKD 26 1.7.1 Protease-activated receptors (PARs) 26 1.7.2 Activated protein C (aPC) 27 2. Aim of the study 32 3. Methods 33 3.1 Reagents 33 3.2 Mice and in vivo interventions 34 3.3 Cell culture and in vitro interventions 35 3.4 Human renal biopsies and urine samples 36 3.5 Glucose uptake assay 41 3.6 In vitro Knockdown 41 3.7 Preparation of activated protein C 42 3.8 Urine collection and processing 42 3.9 p21 ELISA for human urine samples 43 3.10 Albuminuria and in mouse urine samples 43 3.11 Methylation specific PCR (MSP) 43 3.12 Pyrosequencing 44 3.13 DNMT activity assay 44 3.14 Immunoblotting 45 3.15 Reverse transcriptase PCR (RT-PCR) 45 3.16 Quantitative real time PCR (qRT-PCR) 46 3.17 RNA expression profiling 48 3.18 Functional annotation and Pathway analysis 48 3.19 Histology, immunohistochemistry and histological analyses 49 3.20 Immunofluorescence 49 3.21 Senescence associated beta galactosidase (SA-β-gal.) staining 50 3.22 Plasma creatinine and blood urea nitrogen (BUN) 50 3.23 Cell cycle analysis 50 3.24 Statistical Analysis 51 4. Results 52 4.1 Blood glucose normalization does not reverse DKD in experimental models of DM 52 4.2 Identification of genes and pathways associated with hyperglycemic memory 54 4.3 Sustained renal tubular p21 induction in experimental DKD models despite blood glucose normalization 56 4.4 Tubular p21 induction is independent on glomerular damage 58 4.5 Sustained renal tubular p21 expression is associated with induction of senescence 59 4.6. Induction of renal tubular p21 expression in human DKD patients is associated with kidney dysfunction 61 4.7. Sustained p21 expression despite glucose normalization in human DKD patients 63 4.8 aPC reverses glucose induced p21 promoter methylation and sustained p21 expression 64 4.9 Impaired protein C activation increases tubular p21 expression and senescence in vivo 66 4.10 p21 mediates enhanced tubular senescence in aPC-deficient mice 68 4.11 High glucose differentially regulates renal DNMTs expression and activity 70 4.12 Hyperglycemia-induced DNMT1 suppression is part of the hyperglycemic memory 72 4.13 aPC reverses glucose-induced and sustained renal p21 expression via DNMT1 in vivo 74 4.14 aPC reverses glucose-induced and sustained renal tubular senescence via DNMT1 in vivo 76 4.15 aPC requires PAR1 and EPCR to regulate p21 expression 78 4.16 aPC regulates the epigenetically sustained p21 expression independent of its anticoagulant function in vivo 79 4.17 aPC enhances the regenerative capacity of DM kidneys after acute injury by reversing the hyperglycemic memory 81 5. Discussion 85 6. Future perspectives 92 7. Summary of the work 93 8. References 96 Declaration on the independent preparation of the dissertation 104 Curriculum Vitae 106 List of publications 107 Acknowledgement 109

This thesis was about the relationship of the Factor V Leiden mutation and activated protein C resistance. I looked up patients with Leiden mutation and activated protein C resistance. I monitored the frequency of thromboembolism and miscarriages in the personal and family case history of patients. Subsequently, I looked up if other risk factors which affect clinical manifestations in patients with this mutation can be found.