Amplification of EDHF-type vasodilatations in TRPC1-deficient mice

BACKGROUND AND PURPOSE

TRPC1 channels are expressed in the vasculature and are putative candidates for intracellular Ca²⁺ handling. However, little is known about their role in endothelium-dependent vasodilatations including endothelium-derived hyperpolarizing factor (EDHF) vasodilatations, which require activation of Ca²⁺-activated K⁺ channels (K_Ca). To provide molecular information on the role of TRPC1 for K_Ca function and the EDHF signalling complex, we examined endothelium-dependent and independent vasodilatations, K_Ca currents and smooth muscle contractility in TRPC1-deficient mice (TRPC1-/-).

EXPERIMENTAL APPROACH

Vascular responses were studied using pressure/wire myography and intravital microscopy. We performed electrophysiological measurements, and confocal Ca²⁺ imaging for studying K_Ca channel functions and Ca²⁺ sparks.

KEY RESULTS

TRPC1 deficiency in carotid arteries produced a twofold augmentation of TRAM-34- and UCL1684-sensitive EDHF-type vasodilatations and of endothelial hyperpolarization to acetylcholine. NO-mediated vasodilatations were unchanged. TRPC1-/- exhibited enhanced EDHF-type vasodilatations in resistance-sized arterioles in vivo associated with reduced spontaneous tone. Endothelial IK_Ca/SK_Ca-type K_Ca currents, smooth muscle cell Ca²⁺ sparks and associated BK_Ca-mediated spontaneous transient outward currents were unchanged in TRPC1-/-. Smooth muscle contractility induced by receptor-operated Ca²⁺ influx or Ca²⁺ release and endothelium-independent vasodilatations were unaltered in TRPC1-/-. TRPC1-/- exhibited lower systolic blood pressure as determined by tail-cuff blood pressure measurements.

CONCLUSIONS AND IMPLICATIONS

Our data demonstrate that TRPC1 acts as a negative regulator of endothelial K_Ca channel-dependent EDHF-type vasodilatations and thereby contributes to blood pressure regulation. Thus, we propose a specific role of TRPC1 in the EDHF–K_Ca signalling complex and suggest that pharmacological inhibition of TRPC1, by enhancing EDHF vasodilatations, may be a novel strategy for lowering blood pressure.     

Raloxifene protects endothelial cell function against oxidative stress

Background and purpose:

Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress.

Experimental approach:

Changes in isometric tension were measured in rat aortic rings. The content of cyclic GMP in aortic tissue was determined by radioimmunoassay. Phosphorylation of endothelial NOS (eNOS) and Akt was assayed by Western blot analysis.

Key results:

In rings with endothelium, ACh-induced relaxations were attenuated by a ROS-generating reaction (hypoxanthine plus xanthine oxidase, HXXO). The impaired relaxations were ameliorated by acute treatment with raloxifene. HXXO suppressed the ACh-stimulated increase in cyclic GMP levels; this effect was antagonized by raloxifene. The improved endothelial function by raloxifene was abolished by ICI 182,780, and by wortmannin or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Raloxifene also protected endothelial cell function against H₂O₂. Raloxifene increased the phosphorylation of eNOS at Ser-1177 and Akt at Ser-473; this effect was blocked by ICI 182,780. Finally, raloxifene was not directly involved in scavenging ROS, and neither inhibited the activity of xanthine oxidase nor stimulated that of superoxide dismutase.

Conclusion and implications:

Raloxifene is effective against oxidative stress-induced endothelial dysfunction in vitro through an ICI 182,780-sensitive mechanism that involves the increased phosphorylation and activity of Akt and eNOS in rat aortae.     

Vascular reactivity in hypogonadal men is reduced by androgen substitution

The effect of testosterone (T) substitution therapy on blood vessel functions in relation to cardiovascular disease has not been fully elucidated. In 36 newly diagnosed nonsmoking hypogonadal men (37.5 +/- 12.7 yr) endothelium-dependent flow-mediated vasodilatation (FMD; decreased in atherosclerosis) of the brachial artery was assessed before treatment and after 3 months of T substitution therapy (250 mg testosterone enanthate im every 2 wk in 19 men, human chorionic gonadotropin sc twice per week in 17 men). Twenty nonsmoking controls matched for age, low-density lipoprotein cholesterol (LDL-C), body height, and baseline diameter of the artery were selected for repeated measurements from a larger eugonadal control group (n = 113). In hypogonadal men, basal FMD (17.9 +/- 4.5%) was significantly higher than in the large (11.9 +/- 6.4%) and matched control (11.8 +/- 7.1%, both P < 0.001) groups. Grouped multiple linear regression analysis revealed a significant negative association of T levels with FMD within the hypogonadal range, but no significant association was seen within the eugonadal range. During substitution therapy, T levels increased from 5.8 +/- 2.3 to 17.2 +/- 5.1 nmol/liter and FMD decreased significantly to 8.6 +/- 3.1% (P < 0.001, analysis for covariance for repeated measurements including matched controls). LDL-C and advanced age contributed significantly to decrease FMD (P = 0.01, P = 0.04, respectively). Because T substitution adversely affects this important predictor of atherosclerosis, other contributing factors (such as smoking, high blood glucose, and LDL-C) should be eliminated or strictly controlled during treatment of hypogonadal men.     

From the Cover: Protein O-mannosylation is crucial for E-cadherin–mediated cell adhesion

In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell–cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell–cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation–deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell–cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl–conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior.Protein O-mannosylation is a vital protein modification that is evolutionarily conserved across eukaryotes (1). In humans, defects in this modification result in a heterogeneous group of congenital muscular dystrophies (CMDs, α-dystroglycanopathies). The most severe of these disorders, Walker–Warburg syndrome, is characterized by CMD associated with brain malformations of various degrees, ocular abnormalities, and, most often, fatal outcome during the first year of life (2). In contrast, milder disorders such as limb-girdle muscular dystrophy, in which neither the brain nor the eyes are affected, may not present until adulthood (2). The key pathological feature of these diseases is the lack of O-mannosyl glycans on α-dystroglycan (α-DG), an integral component of the dystrophin–glycoprotein complex (2, 3). In the absence of these glycans, binding of α-DG to its extracellular matrix ligands (e.g., laminin) is abolished, and, consequently, basement membranes are fragmented (3–5). In addition to α-DG, O-mannosyl glycans constitute up to 30% of total O-linked carbohydrates in the mammalian brain (6, 7). However, to date only a few other proteins [including CD24 (8), PTPRZ1 (9), neurofascin 186 (10), neurocan, and versican (11)] have been shown to undergo O-mannosylation.Synthesis of O-mannosyl glycans is initiated in the endoplasmic reticulum by the transfer of mannose from dolichol monophosphate-activated mannose (Dol-P-Man) to serine or threonine residues on membrane and secretory proteins (1). In mammals, this reaction is catalyzed by a heteromeric complex of the protein O-mannosyltransferase 1 (POMT1) and 2 (POMT2) (12). We previously showed that Pomt1-null mice display embryonic lethality during postimplantation development, between embryonic day (E)7.5 and E9.5 because of abnormal glycosylation and maturation of α-DG (4). Similarly, knockdown of POMTs in zebrafish and Drosophila melanogaster leads to severe developmental defects that are largely attributable to dysfunctional α-DG (13, 14). Although these animal models have been very helpful in elucidating the role of α-DG–linked O-mannosyl glycans, they have not revealed other biological functions of this protein modification.In both animals and humans, adhesive interactions between neighboring cells are essential for embryogenesis as well as for tissue morphogenesis and renewal (15). Adherens junctions are sites of cell–cell contact where cell-surface receptors of the cadherin family mediate adhesion (16). Classical cadherins are conserved among vertebrates and invertebrates (17). These plasma-membrane glycoproteins share a conserved cytoplasmic domain, a single-pass transmembrane domain, and an ectodomain containing five extracellular cadherin (EC) domains (18). Located on opposing cells, cadherin ectodomains form calcium-dependent homophilic interactions whereby they mediate cell–cell contact (18). The classical cadherin family comprises multiple members, including epithelial cadherin (E-cad, CDH1), neuronal cadherin (N-cad, CDH2), and retinal cadherin (R-cad, CDH4), each of which shows a distinct tissue-specific distribution pattern (16). E-cad plays a critical role in the epithelial–mesenchymal transition during embryogenesis; E-cad–knockout mice die before implantation because of the lack of a functional trophectodermal cell layer at the blastocyst stage (19). Cell–cell adhesion is crucial not only for development but also for tissue morphogenesis and for the invasiveness of human cancer cells (20). Thus, it is particularly important to assess, on a molecular level, the factors that affect this process.In the present study we demonstrate that protein O-mannosylation is essential for the formation of adherens junctions in the preimplantation embryo. Embryos deficient for O-mannosylation die during the morula-to-blastocyst transition because of impaired blastomere adhesion. We identify E-cad as an O-mannosylated glycoprotein and show that this modification is essential for cadherin-mediated cell adhesion. Our identification of functionally relevant O-mannosyl glycans on cadherins is expected to provide further insights into the molecular pathologies of α-dystroglycanopathies.     

Chromosomal instability determines taxane response

Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these “CIN-survival” genes is associated with poor outcome in estrogen receptor–positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.     

Diseases of the salivary glands in infants and adolescents

Background

Current approaches in bone regeneration combine osteoconductive scaffolds with bioactive cytokines like BMP or VEGF. The idea of our in-vitro trial was to apply VEGF₁₆₅ in gradient concentrations to an equine collagen carrier and to study pharmacological and morphological characteristics of the complex in a circulation model.

Methods

Release kinetics of VEGF₁₆₅ complexed in different quantities in a collagen matrix were determined in a circulation model by quantifying protein concentration with ELISA over a period of 5 days. The structural changes of the collagen matrix were assessed with light microscopy, native scanning electron microscopy (SEM) as well as with immuno-gold-labelling technique in scanning and transmission electron microscopy (TEM).

Results

We established a biological half-life for VEGF₁₆₅ of 90 minutes. In a half-logarithmic presentation the VEGF₁₆₅ release showed a linear declining gradient; the release kinetics were not depending on VEGF₁₆₅ concentrations. After 12 hours VEGF release reached a plateau, after 48 hours VEGF₁₆₅ was no longer detectable in the complexes charged with lower doses, but still measurable in the 80 μg sample. At the beginning of the study a smear layer was visible on the surface of the complex. After the wash out of the protein in the first days the natural structure of the collagen appeared and did not change over the test period.

Conclusions

By defining the pharmacological and morphological profile of a cytokine collagen complex in a circulation model our data paves the way for further in-vivo studies where additional biological side effects will have to be considered. VEGF₁₆₅ linked to collagen fibrils shows its improved stability in direct electron microscopic imaging as well as in prolonged release from the matrix. Our in-vitro trial substantiates the position of cytokine collagen complexes as innovative and effective treatment tools in regenerative medicine and and may initiate further clinical research.