Evaluation of beta-galactosidase activity in tissue in the presence of blood

The reporter gene for beta -galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used beta -galactosidase substrates, including o-nitrophenyl-beta -D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG), This may result in false-positive measurements of beta -galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of beta -galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the beta -galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous beta -galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying beta -galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency. Copyright (C) 2000 S. Karger AG, Basel

Vascular dysfunction in COVID-19 patients: update on SARS-CoV-2 infection of endothelial cells and the role of long non-coding RNAs

Although COVID-19 is primarily a respiratory disease, it may affect also the cardiovascular system. COVID-19 patients with cardiovascular disorder (CVD) develop a more severe disease course with a significantly higher mortality rate than non-CVD patients. A common denominator of CVD is the dysfunction of endothelial cells (ECs), increased vascular permeability, endothelial-to-mesenchymal transition, coagulation, and inflammation. It has been assumed that clinical complications in COVID-19 patients suffering from CVD are caused by SARS-CoV-2 infection of ECs through the angiotensin-converting enzyme 2 (ACE2) receptor and the cellular transmembrane protease serine 2 (TMPRSS2) and the consequent dysfunction of the infected vascular cells. Meanwhile, other factors associated with SARS-CoV-2 entry into the host cells have been described, including disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), the C-type lectin CD209L or heparan sulfate proteoglycans (HSPG). Here, we discuss the current data about the putative entry of SARS-CoV-2 into endothelial and smooth muscle cells. Furthermore, we highlight the potential role of long non-coding RNAs (lncRNAs) affecting vascular permeability in CVD, a process that might exacerbate disease in COVID-19 patients

Baroreceptors in the Aortic Arch and Their Potential Role in Aortic Dissection and Aneurysms

The arterial baroreflex is a key autonomic regulator of blood pressure whose dysfunction has been related to several cardiovascular diseases. Changes in blood pressure are sensed by specific mechanosensory proteins, called baroreceptors, particularly located in the outer layer of the carotid sinus and the inner curvature of the aortic arch. The signal is propagated along the afferent nerves to the central nervous system and serves as negative feedback of the heart rate. Despite extensive research, the precise molecular nature of baroreceptors remains elusive. Current knowledge assumes that baroreceptors are ion channels at the nerve endings within the outer layer of the arteries. However, the evidence is based mainly on animal experiments, and the specific types of mechanosensitive receptors responsible for the signal transduction are still unknown. Only a few studies have investigated mechanosensory transmission in the aortic arch. In addition, although aortic dissection, and particularly type A involving the aortic arch, is one of the most life-threatening cardiovascular disorders, there is no knowledge about the impact of aortic dissection on baroreceptor function. In this review, we aim not to highlight the regulation of the heart rate but what mechanical stimuli and what possible ion channels transfer the corresponding signal within the aortic arch, summarizing and updating the current knowledge about baroreceptors, specifically in the aortic arch, and the impact of aortic pathologies on their function

Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo

The potential of two small poly-L-lysines ( sPLLs), low molecular weight sPLL ( LMW-L) containing 7 - 30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer ( GT) with cationic lipid DOCSPER {[}1,3- dioleoyloxy- 2-( N-5-carbamoyl-spermine)-propane] in vascular smooth muscle cells ( SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA ( 50 100 nm). At high ionic strength, large complexes ( 11 mu m) were observed without any significant differences in particle size between lipoplexes ( DOCSPER/ DNA) and lipopolyplexes ( DOCSPER/ sPLL/ DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/ sPLL/ DNA formulations enhanced GT in vitro up to 5- fold, in a porcine model using local periadventitial application up to 1.5- fold. Both sPLLs significantly increased liposome- mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery. Copyright (c) 2007 S. Karger AG, Basel

Alterungsbedingte Gefäßveränderungen am Beispiel der Arteria carotis

Einer der Hauptrisikofaktoren für das Vorliegen einer Karotisstenose und des karotisbedingten Schlaganfalls ist das Lebensalter. Ziel dieses Übersichtsartikels ist die Darstellung des aktuellen Wissensstands über altersbedingte Veränderungen der Gefäße am Beispiel der Karotisstenose.Die Gefäßalterung (vaskuläre Seneszenz) als Abnahme struktureller und funktioneller Eigenschaften der Gefäßwand spielt sich auf verschiedenen Ebenen ab. Auf multizellulärer Ebene kommt es mit zunehmendem Alter hauptsächlich aufgrund von atherosklerotischen Veränderungen der Gefäßwand zu einer Zunahme von Gefäßvolumen und -durchmesser sowie der Intima-Media-Dicke. Auf zellulärer und extrazellulärer Ebene kommt es zur Abnahme von Elastinfasern, glatten Muskelzellen und der Gesamtzellularität sowie zur Zunahme der Lipid‑, Cholesterin- und Kalziumphosphatablagerungen und der Neovaskularisierung. Ursachen der Gefäßalterung auf molekularer Ebene sind insbesondere oxidativer Stress, chronische Entzündungsreaktion, mitochondriale Dysfunktion, epigenetische Veränderungen, Dysregulation der Expression nicht kodierender RNAs (ncRNAs) und die Zunahme der Seneszenz. Der altersbedingte Verlust der Heilungs- und Reparaturfähigkeit des Gewebes macht die Plaques vulnerabler und im Falle der A. carotis anfälliger für ischämische Schlaganfälle.Zunehmende Erkenntnisse über den Einfluss des Alterns auf die Epigenetik und der ncRNAs in atherosklerotischen Plaques kann zukünftig das individuelle Risiko von Patienten genauer quantifizieren und zur Entwicklung zielgerichteter Therapiestrategien beitragen. Weitere Studien sind auf diesem Gebiet jedoch notwendig, um das gesamte Ausmaß der Gefäßalterung und den damit einhergehenden Erkrankungen zu verstehen, damit diesen dann gezielt entgegenwirkt werden kann

Association between carotid diameter and the advanced glycation endproduct Nε-Carboxymethyllysine (CML)

<p>Abstract</p> <p>Background</p> <p>N^ε-Carboxymethyllysine (CML) is the major non-cross linking advanced glycation end product (AGE). CML is elevated in diabetic patients and apparent in atherosclerotic lesions. AGEs are associated with hypertension and arterial stiffness potentially by qualitative changes of elastic fibers. We investigated whether CML affects carotid and aortic properties in normoglycemic subjects.</p> <p>Methods</p> <p>Hundred-two subjects (age 48.2 ± 11.3 years) of the FLEMENGHO study were stratified according to the median of the plasma CML level (200.8 ng/ml; 25^thpercentile: 181.6 ng/ml, 75^thpercentile: 226.1 ng/ml) into "high CML" versus "low CML" as determined by ELISA. Local carotid artery properties, carotid intima media thickness (IMT), aortic pulse wave velocity (PWV), blood pressure and fetuin-A were analyzed. In 26 patients after carotidectomy, CML was visualized using immunohistochemistry.</p> <p>Results</p> <p>According to the CML median, groups were similar for anthropometric and biochemical data. Carotid diameter was enlarged in the "high" CML group (485.7 ± 122.2 versus 421.2 ± 133.2 μm; P < 0.05), in particular in participants with elevated blood pressure and with "high" CML ("low" CML: 377.9 ± 122.2 μm and "high" CML: 514.5 ± 151.6 μm; P < 0.001). CML was associated fetuin-A as marker of vascular inflammation in the whole cohort (r = 0.28; P < 0.01) and with carotid diameter in hypertensive subjects (r = 0.42; P < 0.01). CML level had no effect on aortic stiffness. CML was detected in the subendothelial space of human carotid arteries.</p> <p>Conclusion</p> <p>In normoglycemic subjects CML was associated with carotid diameter without adaptive changes of elastic properties and with fetuin-A as vascular inflammation marker, in particular in subjects with elevated blood pressure. This may suggest qualitative changes of elastic fibers resulting in a defective mechanotransduction, in particular as CML is present in human carotid arteries.</p

MRI plaque imaging reveals high-risk carotid plaques especially in diabetic patients irrespective of the degree of stenosis

<p>Abstract</p> <p>Background</p> <p>Plaque imaging based on magnetic resonance imaging (MRI) represents a new modality for risk assessment in atherosclerosis. It allows classification of carotid plaques in high-risk and low-risk lesion types (I-VIII). Type 2 diabetes mellitus (DM 2) represents a known risk factor for atherosclerosis, but its specific influence on plaque vulnerability is not fully understood. This study investigates whether MRI-plaque imaging can reveal differences in carotid plaque features of diabetic patients compared to nondiabetics.</p> <p>Methods</p> <p>191 patients with moderate to high-grade carotid artery stenosis were enrolled after written informed consent was obtained. Each patient underwent MRI-plaque imaging using a 1.5-T scanner with phased-array carotid coils. The carotid plaques were classified as lesion types I-VIII according to the MRI-modified AHA criteria. For 36 patients histology data was available.</p> <p>Results</p> <p>Eleven patients were excluded because of insufficient MR-image quality. DM 2 was diagnosed in 51 patients (28.3%). Concordance between histology and MRI-classification was 91.7% (33/36) and showed a Cohen's kappa value of 0.81 with a 95% CI of 0.98-1.15. MRI-defined high-risk lesion types were overrepresented in diabetic patients (n = 29; 56.8%). Multiple logistic regression analysis revealed association between DM 2 and MRI-defined high-risk lesion types (OR 2.59; 95% CI [1.15-5.81]), independent of the degree of stenosis.</p> <p>Conclusion</p> <p>DM 2 seems to represent a predictor for the development of vulnerable carotid plaques irrespective of the degree of stenosis and other risk factors. MRI-plaque imaging represents a new tool for risk stratification of diabetic patients.</p> <p>See Commentary: <url>http://www.biomedcentral.com/1741-7015/8/78/abstract</url></p

BCL11A deletions result in fetal hemoglobin persistence and neurodevelopmental alterations

A transition from fetal hemoglobin (HbF) to adult hemoglobin (HbA) normally occurs within a few months after birth. Increased production of HbF after this period of infancy ameliorates clinical symptoms of the major disorders of adult ß-hemoglobin: ß-thalassemia and sickle cell disease. The transcription factor BCL11A silences HbF and has been an attractive therapeutic target for increasing HbF levels; however, it is not clear to what extent BCL11A inhibits HbF production or mediates other developmental functions in humans. Here, we identified and characterized 3 patients with rare microdeletions of 2p15-p16.1 who presented with an autism spectrum disorder and developmental delay. Moreover, these patients all exhibited substantial persistence of HbF but otherwise retained apparently normal hematologic and immunologic function. Of the genes within 2p15-p16.1, only BCL11A was commonly deleted in all of the patients. Evaluation of gene expression data sets from developing and adult human brains revealed that BCL11A expression patterns are similar to other genes associated with neurodevelopmental disorders. Additionally, common SNPs within the second intron of BCL11A are strongly associated with schizophrenia. Together, the study of these rare patients and orthogonal genetic data demonstrates that BCL11A plays a central role in silencing HbF in humans and implicates BCL11A as an important factor for neurodevelopment

5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation

The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors

Serum tissue inhibitor of matrix metalloproteinase-1 levels are associated with mortality in patients with malignant middle cerebral artery infarction

Background: In the last years, circulating matrix metalloproteinases (MMP)-9 levels have been associated with functional outcome in ischemic stroke patients. However the prognostic value of circulating levels of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and MMP-10 in functional outcome of ischemic stroke patients has been scarcely studied. In addition, to our knowledge, serum MMP-9, MMP-10 and TIMP-1 levels in patients with malignant middle cerebral artery infarction (MMCAI) for mortality prediction have not been studied, and these were the objectives of this study. Methods: This was a multicenter, observational and prospective study carried out in six Spanish Intensive Care Units. We included patients with severe MMCAI defined as Glasgow Coma Scale (GCS) lower than 9. We measured circulating levels of MMP-9, MMP-10, TIMP-1, in 50 patients with severe MMCAI at diagnosis and in 50 healthy subjects. Endpoint was 30-day mortality. Results: Patients with severe MMCAI showed higher serum levels of MMP-9 (p = 0.001), MMP-10 (p 239 ng/mL are associated with 30-day mortality (OR = 5.82; 95 % CI = 1.37-24.73; P = 0.02) controlling for GCS and age. The area under the curve for TIMP-1 as predictor of 30-day mortality was 0.81 (95 % CI = 0.67-0.91; P < 0.001). We found an association between circulating levels of TIMP-1 and MMP-10 (rho = 0.45; P = 0.001), plasminogen activator inhibitor (PAI)-1 (rho = 0.53; P < 0.001), and tumor necrosis factor (TNF)-alpha (rho = 0.70; P < 0.001). Conclusions: The most relevant and new findings of our study, were that serum TIMP-1 levels in MMCAI patients were associated with mortality, and could be used as a prognostic biomarker of mortality in MMCAI patients