T-Cadherin Deficiency Is Associated with Increased Blood Pressure after Physical Activity.

T-cadherin is a regulator of blood vessel remodeling and angiogenesis, involved in adiponectin-mediated protective effects in the cardiovascular system and in skeletal muscles. GWAS study has previously demonstrated a SNP in the Cdh13 gene to be associated with hypertension. However, the role of T-cadherin in regulating blood pressure has not been experimentally elucidated. Herein, we generated Cdh13∆Exon3 mice lacking exon 3 in the Cdh13 gene and described their phenotype. Cdh13∆Exon3 mice exhibited normal gross morphology, life expectancy, and breeding capacity. Meanwhile, their body weight was considerably lower than of WT mice. When running on a treadmill, the time spent running and the distance covered by Cdh13∆Exon3 mice was similar to that of WT. The resting blood pressure in Cdh13∆Exon3 mice was slightly higher than in WT, however, upon intensive physical training their systolic blood pressure was significantly elevated. While adiponectin content in the myocardium of Cdh13∆Exon3 and WT mice was within the same range, adiponectin plasma level was 4.37-fold higher in Cdh13∆Exon3 mice. Moreover, intensive physical training augmented the AMPK phosphorylation in the skeletal muscles and myocardium of Cdh13∆Exon3 mice as compared to WT. Our data highlight a critically important role of T-cadherin in regulation of blood pressure and stamina in mice, and may shed light on the pathogenesis of hypertension.

Immature Vascular Smooth Muscle Cells in Healthy Murine Arteries and Atherosclerotic Plaques: Localization and Activity.

The local development of atherosclerotic lesions may, at least partly, be associated with the specific cellular composition of atherosclerosis-prone regions. Previously, it was demonstrated that a small population of immature vascular smooth muscle cells (VSMCs) expressing both CD146 and neuron-glial antigen 2 is postnatally sustained in atherosclerosis-prone sites. We supposed that these cells may be involved in atherogenesis and can continuously respond to angiotensin II, which is an atherogenic factor. Using immunohistochemistry, flow cytometry, wound migration assay xCELLigence system, and calcium imaging, we studied the functional activities of immature VSMCs in vitro and in vivo. According to our data, these cells do not express nestin, CD105, and the leptin receptor. They are localized in atherosclerosis-prone regions, and their number increases with age, from 5.7% to 23%. Immature VSMCs do not migrate to low shear stress areas and atherosclerotic lesions. They also do not have any unique response to angiotensin II. Thus, despite the localization of immature VSMCs and the presence of the link between their number and age, our study did not support the hypothesis that immature VSMCs are directly involved in the formation of atherosclerotic lesions. Additional lineage tracing studies can clarify the fate of these cells during atherogenesis.

Hypercoagulation detected by routine and global laboratory hemostasis assays in patients with infective endocarditis.

BACKGROUND: Coagulation system is heavily involved into the process of infective endocarditis (IE) vegetation formation and can facilitate further embolization. In this study we aimed to assess the coagulation and platelet state in IE implementing a wide range of standard and global laboratory assays. We also aim to determine whether prothrombotic genetic polymorphisms play any role in embolization and mortality in IE patients. METHODS: 37 patients with IE were enrolled into the study. Coagulation was assessed using standard coagulation assays (activated partial thromboplastin time (APTT), prothrombin, fibrinogen, D-dimer concentrations) and integral assays (thromboelastography (TEG) and thrombodynamics (TD)). Platelet functional activity was estimated by flow cytometry. Single nuclear polymorphisms of coagulation system genes were studied. RESULTS: Fibrinogen concentration and fibrinogen-dependent parameters of TEG and TD were increased in patients indicating systemic inflammation. In majority of patients clot growth rate in thrombodynamics was significantly shifted towards hypercoagulation in consistency with D-dimers elevation. However, in some patients prothrombin, thromboelastography and thrombodynamics were shifted towards hypocoagulation. Resting platelets were characterized by glycoprotein IIb-IIIa activation and degranulation. In patients with fatal IE, we observed a significant decrease in fibrinogen and thrombodynamics. In patients with embolism, we observed a significant decrease in the TEG R parameter. No association of embolism or mortality with genetic polymorphisms was found in our cohort. CONCLUSIONS: Our findings suggest that coagulation in patients with infective endocarditis is characterized by general hypercoagulability and platelet pre-activation. Some patients, however, have hypocoagulant coagulation profile, which presumably can indicate progressing of hypercoagulation into consumption coagulopathy.

T-Cadherin and the Ratio of Its Ligands as Predictors of Carotid Atherosclerosis: A Pilot Study.

In the cardiovascular system, atherogenic low-density lipoproteins (LDL) and the protective hormone adiponectin bind to the same receptor, T-cadherin. In this study, we tested the hypothesis that the ratio of circulating LDL to high-molecular weight (HMW) adiponectin could predict the development of atherosclerosis. Using enzyme-linked immunosorbent assay, we measured the level of circulating HMW adiponectin in the blood of donors together with ultrasound measuring of intima-media thickness (IMT) of carotid arteries. Single-nucleotide polymorphisms in the T-cadherin gene were identified using polymerase chain reaction. We found that carotid artery IMT is inversely correlated with the level of HMW in male subjects. We also found that the G allele of rs12444338 SNP in the T-cadherin gene correlates with a lower level of circulating T-cadherin and thinner IMT and therefore could be considered as an atheroprotective genotype. Despite our data, we could not provide direct evidence for the initial study hypothesis. However, we did uncover an important correlation between circulating T-cadherin and thinner carotid IMT.

The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

The membrane of platelets contains at least one uncharacterized glycosylphosphatidylinositol (GPI)-anchored protein according to the literature. Moreover, there is not enough knowledge on the receptor of low-density lipoproteins (LDL) mediating rapid Ca2+ signaling in platelets. Coincidentally, expression of a GPI-anchored protein T-cadherin increases LDL-induced Ca2+ signaling in nucleated cells. Here we showed evidence that supports the hypothesis about the presence of T-cadherin on platelets. The presence of T-cadherin on the surface of platelets and megakaryocytes was proven using antibodies whose specificity was tested on several negative and positive control cells by flow cytometry and confocal microscopy. Using phosphatidylinositol-specific phospholipase C, the presence of glycosylphosphatidylinositol anchor in the platelet T-cadherin form as well as in other known forms was confirmed. We showed by immunoblotting that the significant part of T-cadherin was detected in specific membrane domains (detergent Triton X-114 resistant) and the molecular weight of this newly identified protein was greater than that of T-cadherin from nucleated cells. Nevertheless, polymerase chain reaction data confirmed only the presence of isoform-1 of T-cadherin in platelets and megakaryocytes, which was also present in nucleated cells. We observed the redistribution of this newly identified protein after the activation of platelets, but only further work may explain its functional importance. Thus, our data described T-cadherin with some post-translational modifications as a new GPI-anchored protein on human platelets.

Angiotensin receptor subtypes regulate adipose tissue renewal and remodelling.

Obesity is often associated with high systemic and local renin-angiotensin system (RAS) activity in adipose tissue. Adipose-derived mesenchymal stem/stromal cells (ADSCs), responsible for adipose tissue growth upon high-fat diet, express multiple angiotensin II receptor isoforms, including angiotensin II type 1 receptor (AT1 R), angiotensin II type 2 receptor (AT2 R), Mas and Mas-related G protein-coupled receptor D. Although AT1 R is expressed on most ADSCs, other angiotensin receptors are co-expressed on a small subpopulation of the cells, a phenomenon that results in a complex response pattern. Following AT1 R activation, the effects are transient due to rapid receptor internalisation. This short-lived effect can be prevented by heteromerisation with AT2 R, a particularly important strategy for the regulation of ADSC differentiation and secretory activity. Heteromeric AT2 R might be especially important for the generation of thermogenic beige adipocytes. This review summarises current data regarding the regulation of adipose tissue renewal and particularly ADSC adipogenic differentiation and secretory activity by RAS, with an emphasis on AT2 R and its effects. We reveal a new scheme that implicates AT2 R into the regulation of ADSC hormonal sensitivity.

Optimization of CRISPR/Cas9 Technology to Knock Out Genes of Interest in Aneuploid Cell Lines.

IMPACT STATEMENT: Cell lines represent convenient models to elucidate specific causes of multigenetic and pluricausal diseases, to test breakthrough regenerative technologies. Most commonly used cell lines surpass diploid cells in their accessibility for delivery of large DNA molecules and genome editing, but the main obstacles for obtaining cell models with knockout-targeted protein from aneuploid cells are multiple allele copies and karyotype/phenotype heterogeneity. In the study, we report an original approach to CRISPR-/Cas9-mediated genome modification of aneuploid cell cultures to create functional cell models, achieving highly efficient targeted protein knockout and avoiding "clonal effect" (for the first time to our knowledge).

Unique genetic profile of hereditary hemochromatosis in Russians: high frequency of C282Y mutation in population, but not in patients.

Hereditary hemochromatosis (HH) is a common cause of primary iron overload induced by genetic impairment of iron metabolism. More than 80% of HH patients in populations of European origin are homozygotes for a single mutation C282Y, or compound heterozygotes for C282Y and H63D mutations in the HFE gene. However, in the majority of Asian, African, Australasian, and Amerindian populations, frequencies of C282Y are close to zero. Data on the prevalence of HFE mutations in Russian population and in Russian patients with HH are very limited. In this work, we determined frequencies of C282Y and H63D in ethnical Russians living in the Central European region of Russia. Furthermore, we tested whether homozygocity for C282Y is the major cause of HH in Russians. We found that, in the Russian population, the frequency of C282Y mutation in the HFE gene is relatively high and corresponds to mean European levels. However, in contrast to the majority of European populations, homozygocity for C282Y is found only in a small proportion (5%) of patients with biochemical and clinical signs of HH. These data suggest that either the penetrance of C282Y in Russia is lower than in Western countries, or that a more frequent non-HFE dependent mechanism of primary iron overload dominates in Russian population.