Maria Lyck Hansen

Angiotensin II Regulates microRNA-132/-212 in Hypertensive Rats and Humans

MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are emerging as key regulators in cardiovascular development and disease. MiRNAs are involved in cardiac hypertrophy, heart failure and remodeling following cardiac infarction; however, miRNAs involved in hypertension have not been thoroughly investigated. We have recently reported that specific miRNAs play an integral role in Angiotensin II receptor (AT1R) signaling, especially after activation of the Gαq signaling pathway. Since AT1R blockers are widely used to treat hypertension, we undertook a detailed analysis of potential miRNAs involved in Angiotensin II (AngII) mediated hypertension in rats and hypertensive patients, using miRNA microarray and qPCR analysis. The miR-132 and miR-212 are highly increased in the heart, aortic wall and kidney of rats with hypertension (159 ± 12 mm Hg) and cardiac hypertrophy following chronic AngII infusion. In addition, activation of the endothelin receptor, another Gαq coupled receptor, also increased miR-132 and miR-212. We sought to extend these observations using human samples by reasoning that AT1R blockers may decrease miR-132 and miR-212. We analyzed tissue samples of mammary artery obtained from surplus arterial tissue after coronary bypass operations. Indeed, we found a decrease in expression levels of miR-132 and miR-212 in human arteries from bypass-operated patients treated with AT1R blockers, whereas treatment with β-blockers had no effect. Taken together, these data suggest that miR-132 and miR-212 are involved in AngII induced hypertension, providing a new perspective in hypertensive disease mechanisms.

Global gene expression profiling displays a network of dysregulated genes in non-atherosclerotic arterial tissue from patients with type 2 diabetes

BackgroundGeneralized arterial alterations, such as endothelial dysfunction, medial matrix accumulations, and calcifications are associated with type 2 diabetes (T2D). These changes may render the vessel wall more susceptible to injury; however, the molecular characteristics of such diffuse pre-atherosclerotic changes in diabetes are only superficially known.MethodsTo identify the molecular alterations of the generalized arterial disease in T2D, DNA microarrays were applied to examine gene expression changes in normal-appearing, non-atherosclerotic arterial tissue from 10 diabetic and 11 age-matched non-diabetic men scheduled for a coronary by-pass operation. Gene expression changes were integrated with GO-Elite, GSEA, and Cytoscape to identify significant biological pathways and networks.ResultsGlobal pathway analysis revealed differential expression of gene-sets representing matrix metabolism, triglyceride synthesis, inflammation, insulin signaling, and apoptosis. The network analysis showed a significant cluster of dysregulated genes coding for both intra- and extra-cellular proteins associated with vascular cell functions together with genes related to insulin signaling and matrix remodeling.ConclusionsOur results identify pathways and networks involved in the diffuse vasculopathy present in non-atherosclerotic arterial tissue in patients with T2D and confirmed previously observed mRNA-alterations. These abnormalities may play a role for the arterial response to injury and putatively for the accelerated atherogenesis among patients with diabetes.

Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries From Patients With Type 2 Diabetes Mellitus and Lower Levels Among Metformin Users

Background—The increased risk of cardiovascular diseases in type 2 diabetes mellitus has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes mellitus and moreover hypothesized that metformin intake influences the protein composition. Methods and Results—We analyzed nonatherosclerotic repair arteries gathered at coronary bypass operations from 30 patients with type 2 diabetes mellitus and from 30 age- and sex-matched nondiabetic individuals. Quantitative proteome analysis was performed by isobaric tag for relative and absolute quantitation-labeling and liquid chromatography–mass spectrometry, tandem mass spectrometry analysis on individual arterial samples. The amounts of the basement membrane components, &agr;1-type IV collagen and &agr;2-type IV collagen, &ggr;1-laminin and &bgr;2-laminin, were significantly increased in patients with diabetes mellitus. Moreover, the expressions of basement membrane components and other vascular proteins were significantly lower among metformin users when compared with nonusers. Patients treated with or without metformin had similar levels of hemoglobin A1c, cholesterol, and blood pressure. In addition, quantitative histomorphometry showed increased area fractions of collagen-stainable material in tunica intima and media among patients with diabetes mellitus. Conclusions—The distinct accumulation of arterial basement membrane proteins in type 2 diabetes mellitus discloses a similarity between the diabetic macroangiopathy and microangiopathy and suggests a molecular explanation behind the alterations in vascular remodeling, biomechanical properties, and aneurysm formation described in diabetes mellitus. The lower amounts of basement membrane components in metformin-treated individuals are compatible with the hypothesis of direct beneficial drug effects on the matrix composition in the vasculature.

Proteome Analysis of Human Arterial Tissue Discloses Associations Between the Vascular Content of Small Leucine-Rich Repeat Proteoglycans and Pulse Wave Velocity

Objectives—We hypothesized that arterial stiffness is associated with changes in the arterial protein profile, particularly of extracellular matrix components. We aimed at determining differentially expressed proteins by quantitative proteome analysis in arterial tissue from patients with different degrees of arterial stiffness. Approach and Results—Arterial stiffness, assessed by carotid-femoral pulse wave velocity (PWV), central blood pressure and augmentation index by pulse wave analysis were measured the day before surgery in a group of patients undergoing coronary artery bypass grafting. Protein extracts of well-defined, homogenous, nonatherosclerotic individual samples of the left mammary artery from 10 of these patients with high PWV and 9 with low PWV were compared by quantitative proteome analysis, using tandem mass tag labeling and nano–liquid chromatography mass spectrometry/mass spectrometry. Of 418 quantified proteins, 28 were differentially expressed between the groups with high and low PWV (P<0.05). Three of 7 members of the extracellular matrix family of small leucine-rich repeat proteoglycans displayed significant differences between the 2 groups (P=0.0079; Fisher exact test). Three other ECM proteins were differentially regulated, that is, collagen, type VIII, &agr;-1 and &agr;-2 and collagen, type IV, &agr;-1. Several proteins related to smooth muscle cell function and structure were also found in different amounts between the 2 groups. Conclusions—Changes in the arterial amounts of small leucine-rich proteoglycans, known to be involved in collagen fibrillogenesis, and of some nonfibrillar collagens in combination with alterations in proteins related to functions of the human arterial smooth muscle are associated with arterial stiffness, as determined by PWV.

Elastin Organization in Pig and Cardiovascular Disease Patients' Pericardial Resistance Arteries

Peripheral vascular resistance is increased in essential hypertension. This involves structural changes of resistance arteries and stiffening of the arterial wall, including remodeling of the extracellular matrix. We hypothesized that biopsies of the human parietal pericardium, obtained during coronary artery bypass grafting or cardiac valve replacement surgeries, can serve as a source of resistance arteries for structural research in cardiovascular disease patients. We applied two-photon excitation fluorescence microscopy to study the parietal pericardium and isolated pericardial resistance arteries with a focus on the collagen and elastin components of the extracellular matrix. Initial findings in pig tissue were confirmed in patient biopsies. The microarchitecture of the internal elastic lamina in both the pig and patient pericardial resistance arteries (studied at a transmural pressure of 100 mm Hg) is fiber like, and no prominent external elastic lamina could be observed. This microarchitecture is very different from that in rat mesenteric arteries frequently used for resistance artery research. In conclusion, we add three-dimensional information on the structure of the extracellular matrix in resistance arteries from cardiovascular disease patients and propose further use of patient pericardial resistance arteries for studies of the human microvasculature.

Discordant Diagnoses of Acute Myocardial Infarction due to the Different Use of Assays and Cut-Off Points of Cardiac Troponins

Objectives: Several assays for the measurement of cardiac troponin (cTn) are available, but differences in their analytical performances may affect the diagnosis of acute myocardial infarction (MI). Methods: A survey was conducted at all Danish departments of clinical biochemistry at hospitals receiving patients with suspected acute MI to gather information about the assay and cut-off value used. cTn was measured in blood samples from 574 patients enrolled into the Odense Chest Pain Biobank with 3 different assays: the 4th generation cTnT (cTnT4th), high-sensitivity cTnT (cTnThs; Roche Diagnostics) and cTnI (Abbott Diagnostics). To evaluate concordance, patients were dichotomised according to the 99th percentile value for each assay. Additionally, a cut-off at 50 ng/l for cTnThs was used, as this is the currently employed cut-off point in Denmark. Results: Using a cTnT4th cut-off value of >0.03 µg/l, 130 patients (23%) had potential MI. With the cTnThs assay and cut-off values at 50 versus 14 ng/l, respectively, 136 (24%) versus 301 (52%) patients had potential MI. With the cTnI cut-off point, 205 patients (36%) should be considered as having had an acute MI. Conclusions: The use of different cTn assays and cut-off values may result in a discordant frequency of MI diagnoses. This makes efforts to harmonize cTn assays and cut-off levels mandatory.

Fibulins and their role in cardiovascular biology and disease.

Fibulins are a group of extracellular matrix proteins of which many are present in high amounts in the cardiovascular system. They share common biochemical properties and are often found in relation to basement membranes or elastic fibers. Observations in humans with specific mutations in fibulin genes, together with results from genetically engineered mice and data from human cardiovascular tissue suggest that the fibulin family of proteins play important functional roles in the cardiovascular system. Moreover, fibulin-1 circulates in high concentrations in plasma and may function as a cardiovascular disease marker.

Aortic Valve Stenosis and Atrial Fibrillation Influence Plasma Fibulin-1 Levels in Patients Treated with Coronary Bypass Surgery

Objectives: Aortic valve stenosis (AS) causes cardiac fibrosis and left ventricular hypertrophy, and over time heart failure can occur. To date, a reliable marker to predict progression of AS or the development of heart failure is still lacking. In this study, we addressed the hypothesis that fibulin-1 levels reflect myocardial fibrosis. Methods: Patients undergoing heart surgery at the Odense University were investigated. By 2012 data on outcome were obtained. Results: In 293 patients, plasma fibulin-1 levels were measured. Patients with AS or atrial fibrillation (AF) had significantly higher fibulin-1 levels compared to those with coronary artery disease only (p = 0.005). Patients with preoperatively diagnosed chronic AF had significantly higher levels of fibulin-1 compared to those without (p = 0.004). Plasma fibulin-1 levels showed no relationship to echocardiographic size and had no impact on outcome, death or other adverse events. Conclusion: This study shows that plasma fibulin-1 levels are increased in patients with AS and AF compared to patients with coronary disease only. Our study results suggest fibulin-1, a vascular extracellular matrix (ECM) protein, as a marker of ECM turnover perhaps due to the increased myocardial stretch that is related to pressure overload.

Associations between plasma fibulin-1, pulse wave velocity and diabetes in patients with coronary heart disease

BACKGROUND Diabetes is related to increased risk of cardiovascular disease, and arterial stiffness and its consequences may be the factor connecting the two. Arterial stiffness is often measured by carotid-femoral pulse wave velocity (cf-PWV), but no plasma biomarker reflecting arterial stiffness is available. Fibulin-1 is an extracellular matrix protein, up-regulated in arterial tissue and in plasma in patients with type 2 diabetes. We aimed to evaluate the association between plasma fibulin-1 and arterial stiffness measured by cf PWV in a group of patients with diabetes, and one without, all undergoing coronary artery bypass grafting. METHODS Pulse wave velocity (PWV) and pulse wave analysis including augmentation index (Aix75) was measured in 273 patients, who subsequently underwent a coronary by-pass operation. Plasma samples were drawn and information was gathered on diabetes status, HbA1c, lipids, medication, body mass index, co-morbidities and smoking status. Carotid artery intima-media thickness, as well as estimation of carotid artery plaque burden, and distal blood pressure was also obtained. RESULTS Sixty three patients had diabetes, and this group had significantly higher levels of plasma fibulin-1, PWV and Aix75, compared to the 210 patients who did not have diabetes. In univariate analysis fibulin-1 and pulse wave velocity were not correlated in either group whereas fibulin-1 in patients without diabetes was correlated to Aix75. CONCLUSION Fibulin-1 and arterial stiffness indices are not directly related in patients with cardiac disease, despite the fact that both measures are increased among patients with diabetes.

Abdominal aortic aneurysm, arterial stiffening and the role of the intraluminal thrombus

BACKGROUND Measure of arterial stiffness could be affected by the presence of abdominal aortic aneurysm (AAA) and especially an intraluminal thrombus (ILT). We, therefore, sought to study this possible connection by measuring pulse wave velocity (PWV) and pulse wave analysis (PWA) including augmentation index adjusted to heart rate 75 (Aix75) in patients with AAA ± ILT. PATIENTS AND METHODS PWV and PWA were measured in male patients with AAA from an ongoing Danish AAA screening trial. Information on blood pressure, medications, BMI and smoking status was obtained at inclusion. RESULTS In total, 157 patients were included. Mean age was 73 years. Mean AAA size was 42.2 mm. Fifty-six of the patients had an intraluminal thrombus, and patients with AAA and ILT had a significantly higher Aix75 than patients with AAA but without ILT (Mean = 28.3 ± 1.4 SEM vs. 24.9 ± 0.81, p=0.027), a difference that was also significant when adjusting for AAA size, blood pressure and age. There was no difference in PWV between the groups. CONCLUSIONS Haemodynamic properties of the aorta are affected by the presence of ILT in patients with AAA that is not explained by aortic size. Alternatively, these findings could be explained by associations between ILT and properties of the left ventricle.