Smooth muscle-specific deletion of Ccn2 causes severe aorta malformation and atherosclerosis.

AIMS: Cellular Communication Network Factor 2 (CCN2) is a matricellular protein implicated in fibrotic diseases, with ongoing clinical trials evaluating anti-CCN2-based therapies. By uncovering CCN2 as abundantly expressed in non-diseased artery tissue, this study aimed to investigate the hypothesis that CCN2 plays a pivotal role in maintaining smooth muscle cell (SMC) phenotype and protection against atherosclerosis. METHODS AND RESULTS: Global- and SMC-specific Ccn2 knockout mouse models were employed to demonstrate that Ccn2 deficiency leads to SMC de-differentiation, medial thickening, and aorta elongation under normolipidemic conditions. Inducing hyperlipidemia in both models resulted in severe aorta malformation and a 17-fold increase in atherosclerosis formation. Lipid-rich lesions developed at sites of the vasculature typically protected from atherosclerosis-development by laminar blood flow, covering 90% of aortas, and extending to other vessels, including coronary arteries. Evaluation at earlier time points revealed medial lipid accumulation as a lesion-initiating event. Fluorescently labelled LDL injection followed by confocal microscopy showed increased LDL retention in the medial layer of Ccn2 knockout aortas, likely attributed to marked proteoglycan enrichment of the medial extracellular matrix. Analyses leveraging data from the Athero-Express study cohort indicated relevance of CCN2 in established human lesions, as CCN2 correlated with SMC marker transcripts across 654 transcriptomically profiled carotid plaques. These findings were substantiated through in situ hybridization showing CCN2 expression predominantly in the fibrous cap. CONCLUSIONS: This study identifies CCN2 as a major constituent of the normal artery wall, critical in regulating SMC differentiation and aorta integrity, and possessing a protective role against atherosclerosis development. These findings underscore the need for further investigation into the potential effects of anti-CCN2-based therapies on the vasculature.

MFAP4-Deficiency Aggravates Age-Induced Changes in Resistance Artery Structure, While Ameliorating Hypertension.

BACKGROUND: Abnormalities of resistance arteries may play essential roles in the pathophysiology of aging and hypertension. Deficiency of the vascular extracellular matrix protein MFAP4 (microfibrillar-associated protein 4) has previously been observed as protective against aberrant arterial remodeling. We hypothesized that MFAP4-deficiency would reduce age- and hypertension-dependent arterial changes in extracellular matrix composition and stiffening. METHODS: Mesenteric arteries were isolated from old (20-23 months) littermate Mfap4+/+ and Mfap4-/- mice, and 2-photon excitation microscopy imaging was used to quantify elastin and collagen volumes and dimensions in the vascular wall. Ten-week-old littermate Mfap4+/+ and Mfap4-/- mice were subjected to 20 days of continuous Ang II (angiotensin II) infusion and hypertension was monitored using invasive blood pressure measurements. Arterial stiffness, responses to vascular constrictors, and myogenic tone were monitored using wire- or pressure-myography. Collagen contents were assessed by Western blotting. RESULTS: MFAP4-deficiency significantly increased collagen volume and elastin fragmentation in aged mesenteric arteries without affecting arterial stiffness. MFAP4-deficient mice exhibited reduced diastolic pressure in Ang II-induced hypertension. There was no significant effect of MFAP4-deficiency on mesenteric artery structural remodeling or myogenic tone, although collagen content in mesenteric arteries was tendentially increased in hypertensive Mfap4+/+ mice relative to Mfap4-/- mice. Increased efficacy of vasoconstrictors (phenylephrine, thromboxane) and reduced stiffness were observed in Ang II-treated Mfap4-/- mouse mesenteric arteries in ex vivo myography recordings. CONCLUSIONS: MFAP4-deficiency reduces the elastin/collagen ratio in the aging resistance artery without affecting arterial stiffness. In contrast, MFAP4-deficiency reduces the stiffness of resistance arteries and ameliorates Ang II-induced hypertension.

The angiotensin AT2-receptor agonist compound 21 is an antagonist for the thromboxane TP-receptor - Implications for preclinical studies and future clinical use.

Since the AT2-receptor (AT2R) agonist C21 has structural similarity to the AT1-receptor antagonists Irbesartan and Losartan, which are antagonists not only at the AT1R, but also at thromboxane TP-receptors, we tested the hypothesis that C21 has TP-receptor antagonistic properties as well. Isolated mouse mesenteric arteries from C57BL/6 J and AT2R-knockout mice (AT2R-/y) were mounted in wire myographs, contracted with either phenylephrine or the thromboxane A2 (TXA2) analogue U46619, and the relaxing effect of C21 (0.1 nM - 10 µM) was investigated. The effect of C21 on U46619-induced platelet aggregation was measured by an impedance aggregometer. Direct interaction of C21 with TP-receptors was determined by an ß-arrestin biosensor assay. C21 caused significant, concentration-dependent relaxations in phenylephrine- and U46619-contracted mesenteric arteries from C57BL/6 J mice. The relaxing effect of C21 was absent in phenylephrine-contracted arteries from AT2R-/y mice, whereas it was unchanged in U46619-contracted arteries from AT2R-/y mice. C21 inhibited U46619-stimulated aggregation of human platelets, which was not inhibited by the AT2R-antagonist PD123319. C21 reduced U46619-induced recruitment of ß-arrestin to human thromboxane TP-receptors with a calculated Ki of 3.74 µM. We conclude that in addition to AT2R-agonistic properties, C21 also acts as low-affinity TP-receptor antagonist, and that - depending on the constrictor - both mechanisms can be responsible for C21-induced vasorelaxation. Furthermore, by acting as a TP-receptor antagonist, C21 inhibits platelet aggregation. These findings are important for understanding potential off-target effects of C21 in the preclinical and clinical context and for the interpretation of C21-related myography data in assays with TXA2-analogues as constrictor.

Artificial intelligence assisted compositional analyses of human abdominal aortic aneurysms ex vivo.

Quantification of histological information from excised human abdominal aortic aneurysm (AAA) specimens may provide essential information on the degree of infiltration of inflammatory cells in different regions of the AAA. Such information will support mechanistic insight in AAA pathology and can be linked to clinical measures for further development of AAA treatment regimens. We hypothesize that artificial intelligence can support high throughput analyses of histological sections of excised human AAA. We present an analysis framework based on supervised machine learning. We used TensorFlow and QuPath to determine the overall architecture of the AAA: thrombus, arterial wall, and adventitial loose connective tissue. Within the wall and adventitial zones, the content of collagen, elastin, and specific inflammatory cells was quantified. A deep neural network (DNN) was trained on manually annotated, Weigert stained, tissue sections (14 patients) and validated on images from two other patients. Finally, we applied the method on 95 new patient samples. The DNN was able to segment the sections according to the overall wall architecture with Jaccard coefficients after 65 epocs of 92% for the training and 88% for the validation data set, respectively. Precision and recall both reached 92%. The zone areas were highly variable between patients, as were the outputs on total cell count and elastin/collagen fiber content. The number of specific cells or stained area per zone was deterministically determined. However, combining the masks based on the Weigert stainings, with images of immunostained serial sections requires addition of landmark recognition to the analysis path. The combination of digital pathology, the DNN we developed, and landmark registration will provide a strong tool for future analyses of the histology of excised human AAA. In combination with biomechanical testing and microstructurally motivated mathematical models of AAA remodeling, the method has the potential to be a strong tool to provide mechanistic insight in the disease. In combination with each patients' demographic and clinical profile, the method can be an interesting tool to in supportof a better treatment regime for the patients.

Nitric oxide (NO) synthase but not NO, HNO or H2 O2 mediates endothelium-dependent relaxation of resistance arteries from patients with cardiovascular disease.

BACKGROUND AND PURPOSE: Superoxide anions can reduce the bioavailability and actions of endothelium-derived NO. In human resistance-sized arteries, endothelium-dependent vasodilatation can be mediated by H2 O2 instead of NO. Here, we tested the hypothesis that in resistance arteries from patients with cardiovascular disease, endothelium-dependent vasodilatation is mediated by a reactive oxygen species and not impaired by oxidative stress. EXPERIMENTAL APPROACH: Small arteries were isolated from biopsies of the parietal pericardium of patients undergoing elective cardiothoracic surgery and were studied using immunohistochemical and organ chamber techniques. KEY RESULTS: NO synthases 1, 2 and 3, superoxide dismutase 1 and catalase proteins were observed in the microvascular wall. Relaxing responses to bradykinin were endothelium dependent. During submaximal depolarization-induced contraction, bradykinin-mediated relaxations were inhibited by inhibitors of NO synthases (NOS) and soluble guanylyl cyclase (sGC) but not by scavengers of NO or HNO, inhibitors of cyclooxygenases, neuronal NO synthase, superoxide dismutase or catalase, or by exogenous catalase. During contraction stimulated by endothelin-1, these relaxations were not reduced by any of these interventions except DETCA, which caused a small reduction. CONCLUSION AND IMPLICATIONS: In resistance arteries from patients with cardiovascular disease, endothelium-dependent relaxations seem not to be mediated by NO, HNO or H2 O2 , although NOS and sGC can be involved. These vasodilator responses continue during excessive oxidative stress.

Retinal Vascular Fractal Dimensions and Their Association with Macrovascular Cardiac Disease.

INTRODUCTION: As the only part of the human vasculature, the retina is available for direct, noninvasive inspection. Retinal vascular fractal dimension (DF) is a method to measure the structure of the retinal vascular tree, with higher noninteger values between 1 and 2 representing a more complex and dense retinal vasculature. Retinal vascular structure has been associated with a variety of systemic diseases, and this study examined the association of DF and macrovascular cardiac disease in a case-control design. METHODS: Retinal fundus photos were captured with Topcon TRC-50X in 38 persons that had coronary artery bypass grafting (CABG, cases) and 37 cardiovascular healthy controls. The semiautomatic software VAMPIRE was used to measure retinal DF. RESULTS: Patients with CABG had lower DF of the retinal main venular vessels compared to the control group (1.15 vs. 1.18, p = 0.01). In a multivariable regression model adjusted for gender and age, eyes in the fourth quartile with higher DF were less likely to have CABG compared to patients in the first (OR, 7.20; 95% confidence interval: 1.63-31.86; p = 0.009) and second (OR, 8.25; 95% confidence interval: 1.70-40.01; p = 0.009) quartiles. CONCLUSIONS: This study demonstrates that lower complexity of main venular vessels associates with higher risk of having CABG. The research supports the hypothesis that the retinal vascular structure can be used to assess nonocular macrovascular disease.

LIMK (LIM Kinase) Inhibition Prevents Vasoconstriction- and Hypertension-Induced Arterial Stiffening and Remodeling.

Increased arterial stiffness and vascular remodeling precede and are consequences of hypertension. They also contribute to the development and progression of life-threatening cardiovascular diseases. Yet, there are currently no agents specifically aimed at preventing or treating arterial stiffening and remodeling. Previous research indicates that vascular smooth muscle actin polymerization participates in the initial stages of arterial stiffening and remodeling and that LIMK (LIM kinase) promotes F-actin formation and stabilization via cofilin phosphorylation and consequent inactivation. Herein, we hypothesize that LIMK inhibition is able to prevent vasoconstriction- and hypertension-associated arterial stiffening and inward remodeling. We found that small visceral arteries isolated from hypertensive subjects are stiffer and have greater cofilin phosphorylation than those from nonhypertensives. We also show that LIMK inhibition prevents arterial stiffening and inward remodeling in isolated human small visceral arteries exposed to prolonged vasoconstriction. Using cultured vascular smooth muscle cells, we determined that LIMK inhibition prevents vasoconstrictor agonists from increasing cofilin phosphorylation, F-actin volume, and cell cortex stiffness. We further show that localized LIMK inhibition prevents arteriolar inward remodeling in hypertensive mice. This indicates that hypertension is associated with increased vascular smooth muscle cofilin phosphorylation, cytoskeletal stress fiber formation, and heightened arterial stiffness. Our data further suggest that pharmacological inhibition of LIMK prevents vasoconstriction-induced arterial stiffening, in part, via reductions in vascular smooth muscle F-actin content and cellular stiffness. Accordingly, LIMK inhibition should represent a promising therapeutic means to stop the progression of arterial stiffening and remodeling in hypertension.

Coronary artery bypass surgery independently associates with retinal vascular oxygen saturation.

PURPOSE: The retinal vasculature is the only part of the microcirculation that can be directly studied by non-invasive imaging. Based on the hypothesis that the systemic circulation is reflected in retinal vessels, we investigated if coronary artery bypass grafting (CABG) is related to changes in retinal vascular oxygen saturation (rSatO2 ). METHODS: Retinal metabolism was evaluated by Oxymap T1, which simultaneously captures two retinal images at different wavelengths measuring the retinal arteriolar (raSatO2 ) and venular (rvSatO2 ) oxygen saturation. Three to 4 days after surgery, we measured the median rSatO2 after CABG in 38 patients and in 39 healthy controls (operated for cataract). RESULTS: Coronary artery bypass grafting patients had higher raSatO2 (median ± standard deviation 93.1 ± 6.7% versus 90.5 ± 11.2%, p = 0.001) and rvSatO2 (57.4 ± 8.3% versus 53.5 ± 15.4%, p = 0.048) compared to healthy controls. In multivariable linear regression models, raSatO2 independently associated with CABG (coefficient + 3.6% in CABG patients, p = 0.007), and rvSatO2 correlated with gender (coefficient + 9.4% for females, p = 0.001) and CABG (coefficient + 8.2% in patients with CABG, p = 0.001). CONCLUSIONS: Comparing patients with and without cardiovascular disease, raSatO2 and rvSatO2 positively and independently associated with CABG, suggesting their potential as non-invasive markers for coronary large artery disease.

Implementing collaborative, active learning using peer instructions in pharmacology teaching increases students' learning and thereby exam performance.

Teaching is a highly complex activity that draws on many kinds of knowledge. In this paper, we present our findings on the impact of implementing collaborative, active learning using peer instructions in pharmacology lectures. The hypothesis "implementation of collaborative, active learning increases students' understanding of pharmacological concepts, and thereby exam performance" was proven. Active learning was implemented in lectures on pharmacodynamics (10% of total lectures) in three different pharmacology courses. Teaching methods in the remaining lectures in the courses were unchanged. Prior to each lecture on pharmacodynamics, students were instructed to prepare using questions addressing key concepts in the curriculum that would be covered by the following lecture. In the lectures, students' understanding of these concepts was assessed using anonymous, online polls (ConcepTests). Collaborative learning was supported using peer instructions. Using the anonymous results of 15 written exams with 1097 submissions we show that the percentage of students achieving 50% or more of maximum points per exam question is significantly increased in questions relating to peer instructions lectures (P = 0.029, Odds Ratio (IQR) 1.83 (1.07, 3.15)). The observation is not explained by a general improvement of student performance (P = 0.289, Odds Ratio 1.15 (0.89, 1.51)). Both students' and the teacher have a positive perception of the active learning strategy and the possibility to give and receive instant feedback on students' learning progress. We conclude that implementation of collaborative, active learning using peer instructions, significantly improves students' learning in pharmacology.

Local enrichment of fatty acid-binding protein 4 in the pericardial cavity of cardiovascular disease patients.

BACKGROUND: The pericardial fluid may be representative of the interstitium of the heart. The aim of this study was to discriminate in cardiovascular disease patients between adipocytokines that are produced locally by the heart and those supplied by the circulation. METHODS: Enzyme-linked immunosorbent assays (ELISA) were used to determine levels of N-terminal pro-brain natriuretic peptide (NT-pBNP), fatty acid-binding protein 4 (FABP4), leptin, lipocalin-2, neutrophil elastase, proteinase-3, high sensitivity C-reactive protein (hsCRP) and adiponectin in venous plasma and pericardial fluid harvested during elective cardio-thoracic surgery (n = 132-152). RESULTS: In pericardial fluid compared to plasma, the levels were significantly smaller (p < 0.001) for leptin, lipocalin-2, neutrophil elastase, proteinase-3, hsCRP and adiponectin. For these biomarkers, the ratio of pericardial fluid-to-plasma level ([PF]/[P], median (interquartile range)) was 0.65 (0.47-1.01), 0.78 (0.56-1.09), 0.23 (0.11-0.60), 0.17 (0.09-0.36), 0.14 (0.08-0.35), and 0.25 (0.15-0.34), respectively. In contrast, pericardial fluid was significantly enriched (p < 0.001) in NT-pBNP ([PF]/[P]: 1.9 (1.06-2.73)) and even more so for FABP4 ([PF]/[P]: 3.90 (1.47-9.77)). Moreover, in pericardial fluid, the adipocytokines interrelated all significantly positive and correlated negative to hsCRP, whereas for NT-pBNP only a significantly positive correlation with adiponectin was found. These interrelations were distinct from those in the plasma, as were the correlations of the pericardial biomarkers with patient characteristics compared to plasma. CONCLUSIONS: In cardiovascular disease patients, the pericardial cavity is a distinct adipocytokine microenvironment in which especially FABP4 is mainly derived from the heart.

Extracellular matrix in cardiovascular pathophysiology.

The extracellular matrix (ECM) actively participates in diverse aspects of cardiovascular development and physiology as well as during disease development and progression. ECM roles are determined by its physical and mechanical properties and by its capacity to both release bioactive signals and activate cell signaling pathways. The ECM serves as a storage depot for a wide variety of molecules released in response to injury or with aging. Indeed, there is a plethora of examples describing how cells react to or modify ECM stiffness, how cells initiate intracellular signaling pathways, and how cells respond to the ECM. This Perspectives article reviews the contributions of 21 articles published in the American Journal of Physiology-Heart and Circulatory Physiology in response to a Call for Papers on this topic. Here, we summarize the contributions of these studies focused on the cardiac and vascular ECM. We highlight the translational importance of these studies and conclude that the ECM is a critical component of both the heart and vasculature. Readers are urged to examine and learn from this special Call for Papers.

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

Relaxing Responses to Hydrogen Peroxide and Nitric Oxide in Human Pericardial Resistance Arteries Stimulated with Endothelin-1.

In human pericardial resistance arteries, effects of the endothelium-dependent vasodilator bradykinin are mediated by NO during contraction induced by K+ or the TxA2 analogue U46619 and by H2 O2 during contraction by endothelin-1 (ET-1), respectively. We tested the hypotheses that ET-1 reduces relaxing effects of NO and increases those of H2 O2 in resistance artery smooth muscle of patients with cardiovascular disease. Arterial segments, dissected from the parietal pericardium of 39 cardiothoracic surgery patients, were studied by myography during amplitude-matched contractions induced by K+ , the TXA2 analogue U46619 or ET-1. Effects of the NO donor Na-nitroprusside (SNP) and of exogenous H2 O2 were recorded in the absence and presence of inhibitors of cyclooxygenases, NO synthases and small and intermediate conductance calcium-activated K+ channels. During contractions induced by either of the three stimuli, the potency of SNP did not differ and was not modified by the inhibitors. In vessels contracted with ET-1, the potency of H2 O2 was on average and in terms of interindividual variability considerably larger than in K+ -contracted vessels. Both differences were not statistically significant in the presence of inhibitors of mechanisms of endothelium-dependent vasodilatation. In resistance arteries from patients with cardiovascular disease, ET-1 does not selectively modify smooth muscle relaxing responses to NO or H2 O2 . Furthermore, the candidate endothelium-derived relaxing factor H2 O2 also acts as an endothelium-dependent vasodilator.

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2 ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies.NEW & NOTEWORTHY This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.

Endothelial SIRT1 prevents adverse arterial remodeling by facilitating HERC2-mediated degradation of acetylated LKB1.

Aims-SIRT1 exerts potent activity against cellular senescence and vascular ageing. By decreasing LKB1 protein levels, it promotes the survival and regeneration of endothelial cells. The present study aims to investigate the molecular mechanisms underlying SIRT1-mediated LKB1 degradation for the prevention of vascular ageing.Methods and Results-Co-immunoprecipitation assay demonstrated that SIRT1, via its amino-terminus, binds to the DOC domain of HERC2 [HECT and RLD domain containing E3 ubiquitin protein ligase 2], which then ubiquitinates LKB1 in the nuclear compartment of endothelial cells. Site-directed mutagenesis revealed that acetylation at lysine (K) 64 of LKB1 triggers the formation of SIRT1/HERC2/LKB1 protein complex and subsequent proteasomal degradation. In vitro cellular studies suggested that accumulation of acetylated LKB1 in the nucleus leads to endothelial activation, in turn stimulating the proliferation of vascular smooth muscle cells and the production of extracellular matrix proteins. Chromatin immunoprecipitation quantitative PCR confirmed that acetylated LKB1 interacts with and activates TGFß1 promoter, which is inhibited by SIRT1. Knocking down either SIRT1 or HERC2 results in an increased association of LKB1 with the positive regulatory elements of TGFß1 promoter. In mice without endothelial nitric oxide synthase, selective overexpression of human SIRT1 in endothelium prevents hypertension and age-related adverse arterial remodeling. Lentiviral-mediated knockdown of HERC2 abolishes the beneficial effects of endothelial SIRT1 on both arterial remodeling and arterial blood pressure control.Conclusion-By downregulating acetylated LKB1 protein via HERC2, SIRT1 fine-tunes the crosstalk between endothelial and vascular smooth muscle cells to prevent adverse arterial remodeling and maintain vascular homeostasis.

Biochemical and Bioimaging Evidence of Cholesterol in Acquired Cholesteatoma.

OBJECTIVES: To quantify the barrier sterols and image the lipid structures in the matrix of acquired cholesteatoma and compare the distribution with that found in stratum corneum from normal skin, with the goal to resolve their potential influence on cholesteatoma growth. METHODS: High-performance thin-layer chromatography (HPTLC) was used to achieve a quantitative biochemical determination of the sterols. The intercellular lipids were visualized by Coherent Anti-Stokes Raman scattering (CARS) microscopy, which enables label-free imaging of the lipids in intact tissue samples. RESULTS: The results show that the total lipid content of the cholesteatoma matrix is similar to that of stratum corneum from skin and that the cholesteatoma matrix unquestionably contains cholesterol. The cholesterol content in the cholesteatoma matrix is increased by over 30% (w/w dry weight) compared to the control. The cholesterol sulfate content is below 1% of the total lipids in both the cholesteatoma and the control. Cholesterol ester was reduced by over 30% when compared to the control. CONCLUSIONS: The content of cholesterol in the cholesteatoma matrix is significantly different from that in stratum corneum from skin, and we confirm that the main structure of the cholesteatoma resembles very thick stratum corneum.

Endothelin-1 shifts the mediator of bradykinin-induced relaxation from NO to H2 O2 in resistance arteries from patients with cardiovascular disease.

BACKGROUND AND PURPOSE: We tested the hypothesis that in resistance arteries from cardiovascular disease (CVD) patients, effects of an endothelium-dependent vasodilator depend on the contractile stimulus. EXPERIMENTAL APPROACH: Arteries dissected from parietal pericardium of cardiothoracic surgery patients were studied by myography and imaging techniques. Segments were sub-maximally contracted by K(+) , the TxA2 analogue U46619 or endothelin-1 (ET-1). KEY RESULTS: Relaxing effects of Na-nitroprusside were comparable, but those of bradykinin (BK) were bigger in the presence of ET-1 compared with K(+) or U46619. BK-induced relaxation was (i) abolished by L-NAME in K(+) -contracted arteries, (ii) partly inhibited by L-NAME in the presence of U46619 and (iii) not altered by indomethacin, L-NAME plus inhibitors of small and intermediate conductance calcium-activated K(+) channels, but attenuated by catalase, in ET-1-contracted arteries. This catalase-sensitive relaxation was unaffected by inhibitors of NADPH oxidases or allopurinol. Exogenous H2 O2 caused a larger relaxation of ET-1-induced contractions than those evoked by K(+) or U46619 in the presence of inhibitors of other endothelium-derived relaxing factors. Catalase-sensitive staining of cellular ROS with CellROX Deep Red was significantly increased in the presence of both 1 µM BK and 2 nM ET-1 but not either peptide alone. CONCLUSIONS AND IMPLICATIONS: In resistance arteries from patients with CVD, exogenous ET-1 shifts the mediator of relaxing responses to the endothelium-dependent vasodilator BK from NO to H2 O2 and neither NADPH oxidases, xanthine oxidase nor NOS appear to be involved in this effect. This might have consequences for endothelial dysfunction in conditions where intra-arterial levels of ET-1 are enhanced.

Compromised epidermal barrier stimulates Harderian gland activity and hypertrophy in ACBP-/- mice.

Acyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed adaptation to weaning, including the induction of the hepatic lipogenic and cholesterogenic gene programs. Here we show that ACBP is highly expressed in the Harderian gland, a gland that is located behind the eyeball of rodents and involved in the production of fur lipids and lipids used for lubrication of the eye lid. We show that disruption of the Acbp gene leads to a significant enlargement of this gland with hypertrophy of the acinar cells and increased de novo synthesis of monoalkyl diacylglycerol, the main lipid species produced by the gland. Mice with conditional targeting of the Acbp gene in the epidermis recapitulate this phenotype, whereas generation of an artificial epidermal barrier during gland development reverses the phenotype. Our findings indicate that the Harderian gland is activated by the compromised epidermal barrier as an adaptive and protective mechanism to overcome the barrier defect.