Lukas Zimmerli

Urinary proteomic biomarkers in coronary artery disease

Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis on-line coupled to ESI-TOF-MS enabling characterization of more than 1000 polypeptides per sample. In a first step a “training set” for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients, and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD-specific panel to predict the presence of CAD was evaluated in a blinded study using a “test set.” The signature panel showed sensitivity of 98% (95% confidence interval, 88.7–99.6) and 83% specificity (95% confidence interval, 51.6–97.4). Furthermore the peptide pattern significantly changed toward the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomics analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment.

Urinary proteomic diagnosis of coronary artery disease: identification and clinical validation in 623 individuals.

Objectives We studied the urinary proteome in a total of 623 individuals with and without coronary artery disease (CAD) in order to characterize multiple biomarkers that enable prediction of the presence of CAD. Methods Urine samples were analyzed by capillary electrophoresis coupled online to micro time-of-flight mass spectrometry. Results We defined a pattern of 238 CAD-specific polypeptides from comparison of 586 spot urine samples from 408 individuals. This pattern identified patients with CAD in a blinded cohort of 138 urine samples (71 patients with CAD and 67 healthy individuals) with high sensitivity and specificity (area under the receiver operator characteristic curve 87%, 95% confidence interval 81–92) and was superior to previously developed 15-marker (area under the receiver operator characteristic curve 68%, P < 0.0001) and 17-marker panels (area under the receiver operator characteristic curve 77%, P < 0.0001). The sequences of the discriminatory polypeptides include fragments of alpha-1-antitrypsin, collagen types 1 and 3, granin-like neuroendocrine peptide precursor, membrane-associated progesterone receptor component 1, sodium/potassium-transporting ATPase gamma chain and fibrinogen-alpha chain. Several biomarkers changed significantly toward the healthy signature following 2-year treatment with irbesartan, whereas short-term treatment with irbesartan did not significantly affect the polypeptide pattern. Conclusion Urinary proteomics identifies CAD with high confidence and might also be useful for monitoring the effects of therapeutic interventions.

Urinary collagen fragments are significantly altered in diabetes: a link to pathophysiology.

Background The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D). Methodology/Principal Findings Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92–95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81–94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81–88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed. Conclusions/Significance These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling.

Imported strongyloidosis: A longitudinal analysis of 31 cases

BACKGROUND Attention regarding imported tropical diseases is typically focused on malaria, although other parasitic diseases such as strongyloidosis may also cause serious health problems. The importance of assessing clinical features and of proper diagnosis and treatment is presented on the basis of 31 patients with imported strongyloidosis. METHODS A retrospective analysis was performed regarding patients treated for strongyloidosis in two referral centers in Switzerland from 1998 to 2002. RESULTS Imported strongyloidosis was investigated in 12 travelers and 19 immigrants. The reasons for diagnostic work-up were clinical symptoms in 84% and eosinophilia and screening in each of 22.5%. All patients had a history of travel or residence in endemic areas. Initial therapy was effective in 20 patients, and there was a tendency for a better response to ivermectin compared with the response to other drugs. A significant reduction in blood eosinophil count and serologic antibody titer was observed in patients responding to therapy after an average of 96 and 270 days, respectively. CONCLUSIONS Strongyloidosis must be suspected in travelers and immigrants with skin or abdominal symptoms from regions where Strongyloides stercoralis is highly endemic. The results of this case series confirm that ivermectin is the drug of choice in treating imported strongyloidosis. Response to therapy can be assessed by serology and differential white blood count performed over 6 months after therapy.

Angiogenesis and hypertension: an update

The purpose of this review is to provide a basic understanding of the important relationship between microvascular remodelling, angiogenesis and hypertension, that is, provide an overview of recent experimental and clinical evidence from anti-hypertensive and pro- and anti-angiogenic therapy with respect to hypertension and microvascular structure. Microvascular rarefaction, that is, a loss of terminal arterioles and capillaries, is found in most forms of human and experimental arterial hypertension. This further increases peripheral resistance, and aggravates hypertension and hypertension-induced target organ damage. In some cases with a genetic predisposition, hypertension is preceded by a loss of microvessels. Therefore, new therapies aimed at reversing microvascular rarefaction potentially represent candidate treatments of hypertension. The microvasculature is formed by the continuous balance between de novo angiogenesis and microvascular regression. Imbalanced angiogenesis, in addition to functional shut-off of blood flow, contributes to microvascular rarefaction. Numerous clinical trials assessing anti-angiogenic agents in cancer patients show that this therapy leads to microvascular rarefaction and causes or aggravates hypertension. The development of specific pro-angiogenic treatment to correct hypertension or ischaemic disorders, however, it is still in its infancy. On the other hand, long-term treatment by classic anti-hypertensive therapies that present vasodilator activity can correct for hypertension-associated rarefaction in man.

Vascular stiffness is related to superoxide generation in the vessel wall

Objectives Oxidative stress causes endothelial dysfunction and plays a major role in the pathogenesis of cardiovascular disease. Increased vascular stiffness is an intermediate phenotype in the development of cardiovascular disease. We hypothesized that vascular stiffness is partially determined by oxidative stress. Methods We examined 163 participants out of whom 80 had coronary artery disease. Vascular stiffness was assessed by pulse wave analysis, pulse wave velocity and measurement of aortic compliance by cardiac MRI. Circulating markers of oxidative stress and vascular superoxide generation in saphenous vein were measured. Results After adjustment for age, sex, BMI, heart rate, blood pressure and lipids only carotid-femoral pulse wave velocity and aortic compliance were different between patients and control group. Aortic compliance was reduced (11.4 ± 6.3 vs. 13.9 ± 7.3 ml × 10−3 per mmHg; P = 0.035) and vascular superoxide generation increased (1.01 ± 0.45 vs. 0.76 ± 0.44 nmol/mg per min; P = 0.035) in patients with coronary artery disease compared with those without. In a multiple stepwise regression analysis, aortic compliance was determined by age (P < 0.001) and vascular superoxide production (P = 0.033). CYBA C242T and NOS3 G894T polymorphisms had additive effects on vascular superoxide generation (P = 0.026) and xanthine oxidase activity was increased in patients with CAD (P = 0.043). Genetic factors (P = 0.033) and xanthine oxidase activity (P < 0.001) were also related to aortic compliance. Conclusion By measuring vascular superoxide generation and aortic compliance using cardiac MRI, we demonstrated a functional relationship between oxidative stress and vascular stiffness. Patients identified with high levels of vascular stiffness are most likely to benefit from strategies to reduce vascular oxidative stress.

The pressure of finding human hypertension genes: new tools, old dilemmas.

Researchers in hypertension genetics feel like they are left behind again. It always seems that the ‘other’ complex diseases are ahead in the race. Evolving new technologies in the form of genome-wide arrays and ‘omics’ technologies mean that investigators can now potentially identify many novel disease factors in one large-scale experiment. Hypertension research now faces the challenge of where to go next after the first genome-wide association experiments failed to provide robust candidates. In this review, we contemplate the old dilemma of whether such genes may ever be found; however, we believe advancing technologies and plummeting costs of large-scale experiments will contribute to the identification of novel molecules that underlie essential hypertension.

Rictor in Perivascular Adipose Tissue Controls Vascular Function by Regulating Inflammatory Molecule Expression

Objective—Perivascular adipose tissue (PVAT) wraps blood vessels and modulates vasoreactivity by secretion of vasoactive molecules. Mammalian target of rapamycin complex 2 (mTORC2) has been shown to control inflammation and is expressed in adipose tissue. In this study, we investigated whether adipose-specific deletion of rictor and thereby inactivation of mTORC2 in PVAT may modulate vascular function by increasing inflammation in PVAT. Approach and Results—Rictor, an essential mTORC2 component, was deleted specifically in mouse adipose tissue (rictorad−/−). Phosphorylation of mTORC2 downstream target Akt at Serine 473 was reduced in PVAT from rictorad−/− mice but unaffected in aortic tissue. Ex vivo functional analysis of thoracic aortae revealed increased contractions and impaired dilation in rings with PVAT from rictorad−/− mice. Adipose rictor knockout increased gene expression and protein release of interleukin-6, macrophage inflammatory protein-1&agr;, and tumor necrosis factor-&agr; in PVAT as shown by quantitative real-time polymerase chain reaction and Bioplex analysis for the cytokines in the conditioned media, respectively. Moreover, gene and protein expression of inducible nitric oxide synthase was upregulated without affecting macrophage infiltration in PVAT from rictorad−/− mice. Inhibition of inducible nitric oxide synthase normalized vascular reactivity in aortic rings from rictorad−/− mice with no effect in rictorfl/fl mice. Interestingly, in perivascular and epididymal adipose depots, high-fat diet feeding induced downregulation of rictor gene expression. Conclusions—Here, we identify mTORC2 as a critical regulator of PVAT-directed protection of normal vascular tone. Modulation of mTORC2 activity in adipose tissue may be a potential therapeutic approach for inflammation-related vascular damage.

Proteomics in Gerontology: Current Applications and Future Aspects – A Mini-Review

Background: Aging is closely related to the onset of chronic diseases, such as coronary artery disease, diabetic nephropathy or different types of malignancies, reflecting the demand for novel biomarkers to manage theses diseases. Objective: The analysis of the human proteome for biomarkers has made considerable advances in the last years. Methods: We describe the main technological approaches taken, their advantages and disadvantages. Results: We will review the different clinical sources of material and attempt to highlight the different challenges and approaches associated with these. Age-related changes in the proteome have been described and were found to be highly similar to changes associated with chronic diseases. We will give several examples on the successful application of proteomics in the diagnosis, prognosis and therapy of these chronic diseases. Conclusions: A boost in disease-related proteomic information is expected in the very near future, and will also result in its broad clinical application. However, this view appears to be dependent on the strict adherence to proper technological/analytical parameters, correct statistics, and large databases that allow comparison of datasets provided by different scientists. Clearly, the proteome is by far too complex to be tackled by one laboratory on its own.

Ten-Year Trends in Intoxications and Requests for Emergency Ambulance Service

Abstract Background. Intoxication, whether from alcohol, drugs, or alcohol and drugs in combination, remains a challenging burden on emergency departments. The increasing alcohol consumption among adolescents and young adults, particularly heavy episodic drinking, and the resulting increase in the use of health care resources for alcohol intoxication has been a widely discussed topic. Objective. The aim of our study was to assess and characterize the use of emergency ambulance services that was required as a result of alcohol and drug intoxication in a major metropolitan area. Methods. We conducted a retrospective, longitudinal study over a 10-year period in the greater metropolitan area of Zurich, Switzerland. The study population included intoxicated patients assessed and initially treated by paramedics of the emergency ambulance service. Data were extracted from the ambulance service reports. The primary outcomes measured were trends over time in the numbers and types of intoxication and trends with respect to gender and age distributions of intoxicated patients. Results. An annual increase of about 5% in the number of intoxicated patients requiring emergency ambulance service was observed over the study period. Alcohol use was present in 73% of the cases. The highest number of cases was among patients 25–44 years of age. The greatest increase in the number of cases over time was among patients under 25 years of age. Women comprised 41% of the patients under 25 years of age but only about 35% of older patients. The number of severe injuries and suicide attempts was small, but the number of suicide attempts increased at a higher rate than the overall number of cases of intoxication. There was a significant increase (17.64% per year on average) in the incidence of aggressive behavior toward paramedics from intoxicated patients, although still small in numbers. Conclusions. Our findings suggest two main vulnerable groups: young persons under 25 years of age, with a particular focus on women, having the greatest increase over time, and middle-aged men, having the greatest proportion among all cases observed. Intervention efforts should include a high-risk approach to reduce alcohol-related problems. Key words: alcohol intoxication; substance-related disorders; aggression; injuries; emergency medical services