Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study.

BACKGROUND: Basement membrane (BM) accumulation is a hallmark of micro-vessel disease in diabetes mellitus (DM). We previously reported marked upregulation of BM components in internal thoracic arteries (ITAs) from type 2 DM (T2DM) patients by mass spectrometry. Here, we first sought to determine if BM accumulation is a common feature of different arteries in T2DM, and second, to identify other effects of T2DM on the arterial proteome. METHODS: Human arterial samples collected during heart and vascular surgery from well-characterized patients and stored in the Odense Artery Biobank were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We included ascending thoracic aortas (ATA) (n = 10 (type 2 DM, T2DM) and n = 10 (non-DM)); laser capture micro-dissected plaque- and media compartments from carotid plaques (n = 10 (T2DM) and n = 9 (non-DM)); and media- and adventitia compartments from ITAs (n = 9 (T2DM) and n = 7 (non-DM)). RESULTS: We first extended our previous finding of BM accumulation in arteries from T2DM patients, as 7 of 12 pre-defined BM proteins were significantly upregulated in bulk ATAs consisting of > 90% media. Although less pronounced, BM components tended to be upregulated in the media of ITAs from T2DM patients, but not in the neighbouring adventitia. Overall, we did not detect effects on BM proteins in carotid plaques or in the plaque-associated media. Instead, complement factors, an RNA-binding protein and fibrinogens appeared to be regulated in these tissues from T2DM patients. CONCLUSION: Our results suggest that accumulation of BM proteins is a general phenomenon in the medial layer of non-atherosclerotic arteries in patients with T2DM. Moreover, we identify additional T2DM-associated effects on the arterial proteome, which requires validation in future studies.

Sex differences in aortic valve calcification in severe aortic valve stenosis: association between computer tomography assessed calcification and valvular calcium concentrations.

AIMS: The aims of this study were to investigate the correlation and sex differences between total valve calcium, valve calcium concentration, and aortic valve calcification (AVC) in explanted valves from patients with severe aortic valve stenosis undergoing aortic valve replacement (AVR). METHODS AND RESULTS: Sixty-nine patients with severe aortic stenosis (AS) scheduled for elective AVR underwent echocardiography and cardiac computed tomography (CT) prior to surgery (AVCin vivo) and CT of the explanted aortic valve (AVCex vivo). Explanted valves were prepared in acid solution, sonicated, and analysed with Arsenazo III dye to estimate total valve calcium and valve calcium concentration. Median AVCex vivo was 2082 (1421-2973) AU; mean valve calcium concentration was 1.43 ± 0.42 µmol Ca2+/mg tissue; median total valve calcium 156 (111-255) mg Ca2+, and valve calcium density 52 (35-81) mg/cm2. AVC displayed a strong correlation with total valve calcium (R2 = 0.98, P < 0.001) and a moderate correlation with valve calcium concentration (R2 = 0.62, P < 0.001). Valvular calcium concentration was associated with sex, aortic valve area, and mean gradient. After adjusting for age and estimated glomerular filtration rate, sex and mean gradient remained associated with valve calcium concentrations. CONCLUSION: AVC score provides a strong estimate for total valve calcium but to a lesser degree calcium concentration in the valve tissue of patients with severe AS. Females presented lower valvular calcium concentrations than males irrespective of AS severity, adding evidence and providing support to the important point that sex differences in valvular calcium concentration in AS does not reflect valvular size.

Arterial tissue transcriptional profiles associate with tissue remodeling and cardiovascular phenotype in children with end-stage kidney disease.

Chronic kidney disease (CKD) greatly increases the risk for cardiovascular disease (CVD). However, molecular mechanisms underlying CKD-induced arterial remodeling are largely unknown. We performed a systematic analysis of arterial biopsies from children with stage 5 predialysis CKD participating in the Cardiovascular Comorbidity in Children with Chronic Kidney Disease (4 C) study. For comparison, we studied biopsies from children without CKD, coronary bypass vessels from adults with atherosclerotic coronary heart disease without CKD and aortic sections of subtotally nephrectomized rats. In pediatric CKD patients, gene expression was correlated to the cardiovascular phenotype assessed by surrogate end-points. The arterial calcium content correlated with the intima-media thickness (IMT) of biopsied vessels from pediatric CKD patients, was markedly increased compared to biopsies from children without CKD and comparable to adult coronary bypass patients. Significant transcriptional changes included ECM components, pro-calcifying factors, and physiological calcification inhibitors; most were highly accordant with changes observed in adults with atherosclerosis and in uremic rats. Individual gene expression levels were significantly associated with the left ventricular mass index and carotid intima media thickness. Thus, inflammatory processes (TNF, IL-10), calcification inhibitors (CA2), the Wnt-pathway (FGF-2) and foremost, ECM components (HMGA1, VNN1, VCAN), impact pathobiological responses in arteries from children with CKD.

Abdominal Aortic Aneurysms Growth Is Associated With High Concentrations of Plasma Proteins in the Intraluminal Thrombus and Diseased Arterial Tissue.

Objective- Porosity of the intraluminal thrombus (ILT) is believed to convey biologically active components from the bloodstream toward the aneurismal wall. Accumulation of molecules in the abdominal aortic aneurysmatic tissue may influence vascular protein turnover and regulate abdominal aortic aneurysm growth. We sought to identify proteins with concentrations in the ILT and the abdominal aortic aneurysm wall which associate with aneurysmal expansion rate. Approach and Results- Proteomic analysis by liquid chromatography tandem-mass spectrometry of separated wall and ILT samples was correlated with preoperative aneurysmal growth rate in 24 individuals operated electively for infrarenal abdominal aortic aneurysm. The median preoperative growth rate was 3.8 mm/y (interquartile range, 3) and the mean observational time was 3.3±1.7 years. Plasma components dominated the group of proteins with tissue concentrations, which correlate positively with growth rates ( P<0.001, Fisher exact test, both in the ILT and the wall). In contrast, in the wall and thrombus samples, ECM (extracellular matrix) proteins were significantly more prevalent in the group of proteins with negative correlations to growth rates ( P<0.05, Fisher exact test). Similarly, a long series of proteins, related to cellular functions correlated negatively to growth rates. Conclusions- When the preoperative aneurysmatic growth rate has been high, the concentration of many plasma proteins residing in the ILT and the aneurysmatic tissue is also high, compatible with the hypothesis of increased tissue porosity and accumulation of plasma components as a driver of aneurysm expansion. Moreover, many matrix and cellular proteins which are found in high concentrations in slower-growing aneurysms provides new knowledge about potential treatment targets.

Computed tomography scan based prediction of the vulnerable carotid plaque.

BACKGROUND: Primary to validate a commercial semi-automated computed tomography angiography (CTA) -software for vulnerable plaque detection compared to histology of carotid endarterectomy (CEA) specimens and secondary validating calcifications scores by in vivo CTA with ex vivo non-contrast enhanced computed tomography (NCCT). METHODS: From January 2014 to October 2016 53 patients were included retrospectively, using a cross-sectional design. All patients underwent both CTA and CEA. Sixteen patients had their CEA specimen NCCT scanned. The semi-automated CTA software analyzed carotid stenosis using different HU values defining plaque components. The predictive values of CTA based detection of vulnerable plaques were calculated. Quantification of calcifications on CTA using region of interest (ROI)-function and mathematical equations was done manually, and validated by NCCT of the CEA specimen. RESULTS: The semi-automated CTA software had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 89.1% (95% CI, 73.6% - 96.4%), 31.3% (95% CI, 12.1% - 58.5%), 75% (95% CI, 59.3% - 86.2%) and 55.6% (95% CI, 22.6% - 84.6%). Strong correlation between in vivo CTA and ex vivo NCCT in quantification of calcification was observed, but CTA systematically underestimated calcificationsscore (CALS) with increasing calcification. CONCLUSION: The CTA-software cannot be used in risk assessment of patients, due to poor specificity and NPV. The correlation between in vivo CTA and ex vivo NCCT was strong, proposing it to be used in both scientifically and clinical settings, but studies with larger sample sizes are needed.

Measuring non-polyaminated lipocalin-2 for cardiometabolic risk assessment.

AIMS: Lipocalin-2 is a pro-inflammatory molecule characterized by a highly diversified pattern of expression and structure-functional relationships. In vivo, this molecule exists as multiple variants due to post-translational modifications and/or protein-protein interactions. Lipocalin-2 is modified by polyamination, which enhances the clearance of this protein from the circulation and prevents its excessive accumulation in tissues. On the other hand, animal studies suggest that non-polyaminated lipocalin-2 (npLcn2) plays a causal role in the pathogenesis of obesity-associated medical complications. The present study examined the presence of npLcn2 in samples from healthy volunteers or patients with cardiac abnormalities and evaluated npLcn2 as a biomarker for cardiometabolic risk assessment. METHODS AND RESULTS: Immunoassays were developed to quantify npLcn2 in blood and urine samples collected from 100 volunteers (59 men and 41 women), or venous plasma and pericardial fluid samples obtained from 37 cardiothoracic surgery patients. In healthy volunteers, npLcn2 levels in serum are significantly higher in obese and overweight than in lean subjects. After adjustment for age, gender, smoking, and body mass index (BMI), serum npLcn2 levels are positively correlated with heart rate, circulating triglycerides, high-sensitivity C-reactive protein (hsCRP), and creatinine in plasma. The npLcn2 levels in urine are significantly increased in subjects with metabolic syndrome and positively correlated with BMI, heart rate, circulating triglycerides, and urinary aldosterone. In cardiothoracic surgery patients, the circulating concentrations of npLcn2 are higher (more than two-fold) than those of healthy volunteers and positively correlated with the accumulation of this protein in the pericardial fluid. Heart failure patients exhibit excessive expression and distribution of npLcn2 in mesothelial cells and adipocytes of the parietal pericardium, which are significantly correlated with the elevated plasma levels of npLcn2, total cholesterol, and creatinine. CONCLUSIONS: Quantitative and qualitative evaluation of npLcn2 in human biofluid samples and tissue samples can be applied for risk assessment of healthy individuals and disease management of patients with obesity-related cardiometabolic and renal complications.

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, α-1 and α-2 and collagen, type IV, α-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.

Immunohistochemical visualization of neurons and specific glial cells for stereological application in the porcine neocortex.

The pig is becoming an increasingly used non-primate model in basic experimental studies of human neurological diseases. In spite of the widespread use of immunohistochemistry and cell type specific markers, the application of immunohistochemistry in the pig brain has not been systematically described. Therefore, to facilitate future stereological studies of the neuronal and glial cell populations in experimental neurological diseases in the pig, we established a battery of immunohistochemical protocols for staining of perfusion fixed porcine brain tissue processed as free floating cryostat-, vibratome- or paraffin sections. Antibodies against NeuN, GFAP, S100-protein, MBP, CNPase, CD11b, CD68 (KP1), CD45 and Ki67 were evaluated, and all except CD68 and CD45 resulted in staining of high quality in either type of tissue. Each staining was evaluated with respect to specificity and sensitivity in identification of the individual cells, and for penetration of the staining and maintenance of section thickness above 25 microm, necessary for stereological cell counting. In the cases of NeuN, CNPase, CD11b and Ki67 the staining met the demands to be applicable in stereological analyses using the optical disector. In conclusion, all protocols will be applicable in studies of pathological and neurochemical changes in the porcine brain, and a few protocols applicable for stereology.