Mycobacterium tuberculosis ClpC1: A potential target for tuberculosis drug discovery

Master'sJOINT M.SC. IN INFECTIOUS DISEASES, VACCINOLOGY AND DRUG DISCOVER

High density lipoprotein from patients with coronary disease exerts altered endothelial effects : role of proteome remodeling

Epidemiological studies have strongly suggested that reduced plasma levels of HDL cholesterol are associated with an increased risk of coronary artery disease. Raising HDL Cholesterol is therefore being examined as a potentially important therapeutic strategy. However, recent genetic data and clinical trials have been inconclusive on the beneficial effects of raising HDL cholesterol levels. Clinical trials using the HDL cholesterol raising agents torcetrapib, dalcetrapib and niacin have shown that no significant reduction of cardiovascular events was observed in patients with coronary disease. These recent failures in treatments that raise HDL cholesterol levels have cast doubt on whether HDL plays a truly protective role in cardiovascular disease. In recent years, various biological functions of HDL have been identified, that may account for the ability of HDL to protect against atherosclerosis. Endothelial dysfunction is suggested to play a critical role in development and progression of atherosclerosis and recent studies have suggested that HDL exerts direct endothelial-protective effects, such as stimulation of endothelial production of the anti-atherogenic molecule nitric oxide, anti-inflammatory, anti-apoptotic, and anti-thrombotic effects. However, accumulating evidence suggests that the vascular effects of HDL may be highly heterogenous and vasoprotective properties of HDL may be impaired in patients with inflammatory or cardiovascular disease. It is therefore essential to elucidate the mechanisms leading to impaired vascular effects of HDL in coronary artery disease and to understand the importance of raising HDL with atheroprotective quality. The first aim of this thesis was to investigate the effects of HDL isolated from patients with coronary artery disease (CAD) on endothelial function and to characterize the mechanisms leading to impaired vascular effects of HDL in patients with CAD, in particular the effects of HDL on endothelial apoptotic signaling pathways. The second aim of my thesis was to optimize cell-based assays that allow for high-throughput assessment of the endothelial effects of HDL. Finally, in order to elucidate the mechanisms of action of HDL and its altered vascular effects in cardiovascular diseases, there is a need to characterize HDL cargo. The third aim of my thesis was to use targeted proteomics to investigate changes in the protein compositions of HDL that may modulate its function in coronary artery disease. In order to investigate the effects of HDL on endothelial apoptosis signaling and its alterations in coronary disease, I have isolated HDL from patients with CAD and characterized its mechanisms of action. In contrast to HDL from healthy subjects, HDL isolated from patients with CAD failed to inhibit endothelial cell apoptosis in vitro and in apoE-deficient-mice in vivo. Instead, HDL isolated from these patients stimulated endothelial pro-apoptotic pathways, in particular p38-MAPK-mediated activation of the pro-apoptotic Bcl- 2-protein tBid. This study further suggests that differences in the proteome of HDL from patients with CAD, in particular reduced HDL-associated clusterin and increased HDL-associated apoC-III, play an important role for altered activation of endothelial anti- and pro-apoptotic signaling pathways. I have optimized cell-based assays for the assessment of HDL function, in particular the capacity of HDL to inhibit TNF-α-induced VCAM-1 expression, to reduce active caspase-3 activation and to stimulate nitric oxide production. These assays were used to evaluate the impact of cholesteryl ester transfer protein (CETP) inhibitor treatment on the endothelial effects of HDL. Treatment with dalcetrapib failed to restore HDL functions to the level of healthy subjects, which may explain, at least in part, the lack of benefit on endothelial function seen in the dal-VESSEL trial. Interestingly, despite the failure of torcetrapib in clinical trial resulting in increased cardiovascular mortality, no impairment in HDL functions was observed following torcetrapib treatment in this study. This finding may explain the lack of improvement in the quality of HDL following torcetrapib treatment, however the adverse effects observed in the clinical trial of torcetrapib were likely caused by off-target side effects, which may have been due to direct deleterious actions of torcetrapib on endothelial function, independent of the HDL cholesterol raising effect. Last but not least, using quantitative proteomics analysis with selected reaction monitoring (SRM), it was observed that HDL from patients with CAD has significantly altered protein compositions as compared to HDL from healthy subjects, further supporting our earlier observations. Notably, significant differences were observed in the HDL proteome between patients with CAD who died from cardiovascular events versus patients who did not develop any events during the entire follow-up study period. This finding may identify potential markers in determining patients at risk of developing future cardiovascular events. More importantly, some of the proteins identified will allow better understanding of the mechanisms of action of HDL and its alterations in cardiovascular disease. Of note, some of the proteins identified to be differentially regulated are not related to lipoprotein metabolism, indicating that HDL physiology and its pathophysiology in CAD is beyond simply regulation of lipid metabolism but also other metabolic processes such as innate immunity and regulation of oxidative stress. In epidemiologischen Studien waren verringerte Plasmaspiegel des HDL-Cholesterols mit einem erhöhten Risiko für koronare Herzerkrankung (KHK) assoziiert. Daher wird derzeit untersucht, ob die Steigerung der HDL-Cholesterolspiegels im Blut eine erfolgreiche therapeutische Strategie bei KHK darstellt. Jedoch lieferten aktuelle genetische Untersuchungen und klinische Studien widersprüchliche Daten zum Effekt einer Steigerung der HDL-Spiegel. Klinische Studien, bei denen die HDL-steigernden Substanzen Torcetrapib, Dalcetrapib und Niacin eingesetzt wurden, konnte bei Patienten mit KHK keine signifikante Verringerung kardiovaskulärer Ereignisse beobachtet werden. Diese Studienergebnisse führten zu Zweifeln an der postulierten protektiven Wirkung einer HDL-Steigerung bei KHK. In den vergangenen Jahren wurden verschiedene biologische Funktionen des HDL-Cholesterols identifiziert, welche dessen atheroprotektive Wirksamkeit beeinflussen könnten. Besonders der endothelialen Dysfunktion wird eine wichtige Rolle in der Entstehung und Entwicklung der Atherosklerose zugeschrieben und von aktuellen Studienergebnissen wurde eine endothel-protektive Wirkung des HDL-Cholesterols abgeleitet, beispielsweise die Produktion des anti-atherogenen Stickstoffmonoxids durch Endothelzellen, sowie weitere inflammationshemmende, überlebensfördernde und thrombosehemmende Eigenschaften. Allerdings deuten die Ergebnisse mehrerer Studien darauf hin dass die Eigenschaften des isolierten HDLs sehr heterogen und abhängig vom Gesundheitszustand des Spenders sind. Die gefässprotektiven Eigenschaften des HDL könnten bei Patienten mit inflammatorischen oder kardiovaskulären Erkrankungen verringert sein. Es ist daher wichtig, die Mechanismen zu verstehen, die zu einer Einschränkung der gefässschützenden Eigenschaften des HDL bei Patienten mit KHK führen und die Bedeutung einer Erhöhung speziell von HDL mit gefässschützenden Eigenschaften zu verstehen. Das Ziel dieser Dissertation war daher zunächst, die Effekte von HDL von Patienten mit KHK auf die Endothelfunktion zu untersuchen und die Mechanismen zu charakterisieren, welche die vaskulären Eigenschaften des HDLs bei Patienten mit KHK einschränken, speziell die Effekte des HDLs auf apoptose- relevante Signalwege in Endothelzellen. Das zweite Ziel war, zellbasierte Tests für die Hochdurchsatz- Analyse der endothelialen Effekte des HDLs zu optimieren. Weiterhin war es wichtig, die Proteinbeladung des HDLs zu charakterisieren, um die Wirkmechanismen des HDLs und seine veränderten vaskulären Effekte bei kardiovaskulären Erkrankungen besser zu verstehen. Das dritte Ziel der Dissertation war daher, die gezielte Proteomanalyse einzusetzen, um Veränderungen in der Proteinzusammensetzung des HDLs zu identifizieren, die dessen Funktion bei Patienten mit KHK verändern könnten. Für die Charakterisierung der molekularen Effekte des HDL auf zelluläre Signalwege zur Steuerung der endothelialen Apoptose und deren Änderung bei KHK habe ich zunächst HDL von Patienten mit KHK sowie von gesunden Probanden isoliert und dessen molekulare Effekte untersucht. Im Vergleich zu gesundem HDL war HDL von Patienten mit KHK nicht in der Lage, die Apoptose von Endothelzellen in vitro und in vivo in ApolipoproteinE-defizienten Mäusen zu verhindern. Stattdessen stimulierte das HDL von KHK-Patienten pro-apoptotische Signalwege in Endothelzellen, wie die Aktivierung des pro-apoptotischen Proteins tBid, eines Mitglieds der Bcl-2-Proteinfamilie, durch die Kinase p38-MAPK. Die Studienergebnisse deuten weiterhin darauf hin, dass Unterschiede im Proteom des HDL von KHK-Patienten, einschliesslich einer Verringerung von HDL-assoziiertem Clusterin und einer Erhöhung von HDL-assoziiertem ApoC-III, eine wichtige Rolle bei der Modulierung von endothelialen pro- und anti-apoptotischen Signalwegen spielen. Ich habe zellbasierte Untersuchungsmethoden der HDL-Funktion für Hochdurchsatzanalysen optimiert, speziell die Bestimmung der Fähigkeit des HDLs, die TNF-α-induzierte Hochregulation der VCAM-1- Expression und die Aktivierung der Caspase-3 zu verringern, sowie die Produktion von Stickstoffmonoxid zu induzieren. Diese Methoden wurden verwendet, um den Einfluss von Inhibitoren des Cholesterylester- Transferproteins (CETP) auf die endothelialen Effekte des HDLs zu testen. Die Behandlung mit Dalcetrapib bei KHK-Patienten konnte die HDL-Funktion nicht auf die Werte des HDLs von gesunden Probanden anheben. Dies könnte, zummindest teilweise, erklären warum in der “Dal-VESSEL”-Studie keine Verbesserung der Endothelfunktion beobachtet werden konnte. Interessant ist, dass wir trotz des Versagens von Torcetrapib in klinischen Studien, bei denen eine erhöhte Mortalität beschrieben wurde, keine Verschlechterung der HDL-Funktion beobachteten. Dies mag erklären, warum keine Verbesserung der HDL- Qualität durch Torcetrapib erreicht werden konnte. Die beobachteten unerwünschten Ereignisse, die in der Torcetrapib-Studie beobachtet wurden, wurden möglicherweise durch Nebenwirkungen des Medikaments hervorgerufen, beispielsweise durch direkte, HDL-unabhängige Effekte des Torcetrapibs auf die Endothelfunktion. Im letzten Teil der Dissertation konnte mithilfe der quantitativen Proteomanalyse, bei der die Technik „selected reaction monitoring (SRM)“ eingesetzt wurde, gefunden werden, dass sich die Proteinzusammensetzung des HDLs von Patienten mit KHK signifikant vom HDL gesunder Probanden unterscheidet. Dies unterstützt zusätzlich unsere früheren Beobachtungen. Von besonderer Bedeutung ist die Identifizierung von Unterschieden in der HDL-Zusammensetzung von Patienten mit KHK, die später an einem kardiovaskulären Ereignis verstarben, im Vergleich zu Patienten, welche während des follow-up- Zeitraums kein Ereignis erlitten hatten. Diese Beobachtung könnte in der Zukunft zur Identifizierung von Markern dienen, um Patienten zu erkennen, die ein erhöhtes Risiko haben, ein kardiovaskuläres Ereignis zu erleiden. Weiterhin ermöglichen einige der identifizierten Proteine ein besseres Verständnis der Wirkmechanismen des HDLs und seiner Veränderungen bei kardiovaskulären Erkrankungen. In diesem Zusammenhang ist von besonderem Interesse, dass für einige der verändert vorhandenen Proteine bislang keine Rolle im Lipoproteinmetabolismus bekannt war. Dies legt den Schluss nahe, dass die physiologische Rolle des HDL, sowie seine pathophysiologischen Veränderungen bei KHK , sich über die einfache Regulation des Lipidmetabolismus erstreckt und weitere Prozesse, beispielsweise die unspezifische Immunabwehr und die Regulation des oxidativen Stresses beeinflusst

Altered activation of endothelial anti- and proapoptotic pathways by high-density lipoprotein from patients with coronary artery disease: role of high-density lipoprotein-proteome remodeling

BACKGROUND: Endothelial dysfunction and injury are thought to play an important role in progression of coronary-artery-disease (CAD). High-density-lipoprotein from healthy subjects (HDLHealthy) has been proposed to exert endothelial anti-apoptotic effects that may represent an important anti-atherogenic property of the lipoprotein. The present study therefore aimed to compare effects of HDLCAD and HDLHealthy on activation of endothelial anti- and pro-apoptotic pathways and to determine which changes of the lipoprotein are relevant for these processes. METHODS AND RESULTS: HDL was isolated from patients with stable CAD (HDLsCAD), an acute coronary syndrome (HDLACS) and healthy subjects. HDLHealthy induced expression of the endothelial anti-apoptotic Bcl-2 protein Bcl-xL and reduced endothelial cell apoptosis in vitro and in apoE-deficient-mice in vivo. In contrast, HDLsCAD and HDLACS did not inhibit endothelial apoptosis, failed to activate endothelial Bcl-xL and stimulated endothelial pro-apoptotic pathways, in particular p38-MAPK-mediated activation of the pro-apoptotic Bcl-2-protein tBid. Endothelial anti-apoptotic effects of HDLHealthy were observed after inhibition of endothelial nitric-oxide-synthase and after delipidation, but not completely mimicked by apoA-I or reconstituted HDL, suggesting an important role of the HDL-proteome. HDL proteomics analyses and subsequent validations and functional characterizations suggested a reduced clusterin- and increased apoC-III-content of HDLsCAD and HDLACS as mechanisms leading to altered effects on endothelial apoptosis. CONCLUSIONS: The present study demonstrates for the first time that HDLCAD does not activate endothelial anti-apoptotic pathways, but rather stimulates potential endothelial pro-apoptotic pathways. HDL-proteome remodeling plays an important role for these altered functional properties of HDL. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in coronary disease

Inhibition of Aerobic Glycolysis Attenuates Disease Progression in Polycystic Kidney Disease

Dysregulated signaling cascades alter energy metabolism and promote cell proliferation and cyst expansion in polycystic kidney disease (PKD). Here we tested whether metabolic reprogramming towards aerobic glycolysis ("Warburg effect") plays a pathogenic role in male heterozygous Han:SPRD rats (Cy/+), a chronic progressive model of PKD. Using microarray analysis and qPCR, we found an upregulation of genes involved in glycolysis (Hk1, Hk2, Ldha) and a downregulation of genes involved in gluconeogenesis (G6pc, Lbp1) in cystic kidneys of Cy/+ rats compared with wild-type (+/+) rats. We then tested the effect of inhibiting glycolysis with 2-deoxyglucose (2DG) on renal functional loss and cyst progression in 5-week-old male Cy/+ rats. Treatment with 2DG (500 mg/kg/day) for 5 weeks resulted in significantly lower kidney weights (-27%) and 2-kidney/total-body-weight ratios (-20%) and decreased renal cyst index (-48%) compared with vehicle treatment. Cy/+ rats treated with 2DG also showed higher clearances of creatinine (1.98±0.67 vs 1.41±0.37 ml/min), BUN (0.69±0.26 vs 0.40±0.10 ml/min) and uric acid (0.38±0.20 vs 0.21±0.10 ml/min), and reduced albuminuria. Immunoblotting analysis of kidney tissues harvested from 2DG-treated Cy/+ rats showed increased phosphorylation of AMPK-α, a negative regulator of mTOR, and restoration of ERK signaling. Assessment of Ki-67 staining indicated that 2DG limits cyst progression through inhibition of epithelial cell proliferation. Taken together, our results show that targeting the glycolytic pathway may represent a promising therapeutic strategy to control cyst growth in PKD

Effect of Sodium-Glucose Cotransport Inhibition on Polycystic Kidney Disease Progression in PCK Rats.

The sodium-glucose-cotransporter-2 (SGLT2) inhibitor dapagliflozin (DAPA) induces glucosuria and osmotic diuresis via inhibition of renal glucose reabsorption. Since increased diuresis retards the progression of polycystic kidney disease (PKD), we investigated the effect of DAPA in the PCK rat model of PKD. DAPA (10 mg/kg/d) or vehicle was administered by gavage to 6 week old male PCK rats (n=9 per group). Renal function, albuminuria, kidney weight and cyst volume were assessed after 6 weeks of treatment. Treatment with DAPA markedly increased glucose excretion (23.6 ± 4.3 vs 0.3 ± 0.1 mmol/d) and urine output (57.3 ± 6.8 vs 19.3 ± 0.8 ml/d). DAPA-treated PCK rats had higher clearances for creatinine (3.1 ± 0.1 vs 2.6 ± 0.2 ml/min) and BUN (1.7 ± 0.1 vs 1.2 ± 0.1 ml/min) after 3 weeks, and developed a 4-fold increase in albuminuria. Ultrasound imaging and histological analysis revealed a higher cyst volume and a 23% higher total kidney weight after 6 weeks of DAPA treatment. At week 6 the renal cAMP content was similar between DAPA and vehicle, and staining for Ki67 did not reveal an increase in cell proliferation. In conclusion, the inhibition of glucose reabsorption with the SGLT2-specific inhibitor DAPA caused osmotic diuresis, hyperfiltration, albuminuria and an increase in cyst volume in PCK rats. The mechanisms which link glucosuria to hyperfiltration, albuminuria and enhanced cyst volume in PCK rats remain to be elucidated

Characterization of levels and cellular transfer of circulating lipoprotein-bound microRNAs

OBJECTIVE: MicroRNAs are important intracellular regulators of gene expression, but also circulate in the blood being protected by extracellular vesicles, proteins, or high-density lipoprotein (HDL). Here, we evaluate the regulation and potential function of HDL- and low-density lipoprotein-bound miRs isolated from healthy subjects and patients with coronary artery disease. APPROACH AND RESULTS: HDL-bound miRs with known effects in the cardiovascular system were analyzed in HDL isolated from healthy subjects (n=10), patients with stable coronary artery disease (n=10), and patients with an acute coronary syndrome (n=10). In HDL from healthy subjects, miR-223 was detected at concentrations >10 000 copies/µg HDL, and miR-126 and miR-92a at about 3000 copies/µg HDL. Concentrations of most miRs were substantially higher in HDL as compared with low-density lipoprotein. However, HDL-bound miR-223 contributed to only 8% of the total circulating miRs. The signatures of miRs varied only slightly in HDL derived from patients with coronary artery disease. We did not observe a significant uptake of HDL-bound miRs into endothelial cells, smooth muscle cells, or peripheral blood mononuclear cells. However, patient-derived HDL transiently reduced miR expression particularly when incubated with smooth muscle and peripheral blood mononuclear cells. CONCLUSIONS: Circulating miRs are detected in HDL and to a lesser extent in low-density lipoprotein, and the miR-signatures are only slightly altered in patients with coronary artery disease. Lipoprotein-bound miRs were not efficiently delivered to endothelial, smooth muscle, and peripheral blood mononuclear cells suggesting that the lipoprotein-associated pool of miRs is not regulating the function of the studied cells in vitro

Inhibition of Sodium-GlucoseCotransporter 2 with dapagliflozin in Han: SPRD rats with polycystic kidney disease

BACKGROUND/AIMS Dapagliflozin (DAPA) is a selective inhibitor of the sodium-glucose cotransporter 2 (SGLT2) which induces glucosuria and osmotic diuresis. The therapeutic effect of DAPA in progressing stages of polycystic kidney disease (PKD) has not been studied. METHODS We examined the effect of DAPA in the Han: SPRD rat model of PKD. DAPA (10 mg/kg/day) or vehicle (VEH) was administered orally via gavage to 5 week old male Han: SPRD (Cy/+) or control (+/+) rats (n = 8-9 per group) for 5 weeks. Blood and urine were collected at baseline and after 2.5 and 5 weeks of treatment to assess renal function and albuminuria. At the end of the treatment, rats were sacrificed and kidneys were excised for histological analysis. RESULTS After 5 weeks of treatment, DAPA-treated Cy/+ and +/+ rats exhibited significantly higher glucosuria, water intake and urine output than VEH-treated rats. DAPA-treated Cy/+ rats also exhibited significantly higher clearances for creatinine and BUN and less albuminuria than VEH-treated Cy/+ rats. DAPA treatment for 5 weeks resulted in a significant increase of the kidney weight in Cy/+ rats but no change in cyst growth. The degree of tubular epithelial cell proliferation, macrophage infiltration and interstitial fibrosis was also similar in DAPA-and VEH-treated Cy/+ rats. CONCLUSION The induction of glucosuria with the SGLT2-specific inhibitor DAPA was associated with improved renal function and decreased albuminuria, but had no effect on cyst growth in Cy/+ rats. Overall the beneficial effects of DAPA in this PKD model were weaker than the previously described effects of the combined SGLT1/2 inhibitor phlorizin

Dysregulation of the glycolysis and gluconeogenesis pathways in rat polycystic kidney disease.

<p>(A) Microarray analysis showing differential expression of genes coding for glycolysis and gluconeogenesis enzymes in Han:SPRD Cy/+ and wild-type +/+ kidneys. Upregulated genes are shown in red, and downregulated genes are shown in green. (B) Schematic diagram showing the glycolysis/gluconeogenesis cascades. In red, upregulated genes; green, downregulated genes; black, genes unchanged in kidneys from Cy/+ rats compared with wild-type +/+ kidneys. (C) Real-time quantitative PCR analysis of genes coding for key enzymes involved in glycolysis/gluconeogenesis in kidneys from Cy/+ rats and +/+ rats. (D) Real-time quantitative PCR analysis of the hexokinase-1 (Hk1) and hexokinase-2 (Hk2) genes in primary cell cultures of human ADPKD and control NHK cells. The expression levels of β-actin were used as a housekeeping gene.</p

Schematic diagram showing the interplay of various signaling pathways involved in the regulation of glycolysis in polycystic kidney disease.

<p>Schematic diagram showing the interplay of various signaling pathways involved in the regulation of glycolysis in polycystic kidney disease.</p

Effect of 2DG treatment on parameters of renal function.

<p>Measurement of (A) creatinine clearance, (B) BUN clearance, and (C) uric acid clearance, in +/+ and Cy/+ rats upon treatment with 2DG or vehicle at baseline, 2.5 weeks and 5 weeks. Black, +/+ treated with vehicle; blue, +/+ treated with 2DG; red, Cy/+ treated with vehicle; green, Cy/+ treated with 2DG. *P<0.05, **P<0.01 when comparing Cy/+ 2DG and Cy/+ vehicle at each time point. ^#P<0.05, ^{# #} P<0.01 when comparing Cy/+ and +/+ group. (D) Urine albumin excretion in Cy/+ and +/+ rats after 5-week treatment with 2DG or vehicle. (E) SDS-polyacrylamide gel electrophoresis of urine samples from Cy/+ and +/+ rats after 5-week treatment with 2DG or vehicle. (F) Lactate content in the kidneys of Cy/+ and +/+ rats after treatment with 2DG or vehicle.</p