Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate haemostasis of victims through effects on platelets, vascular endothelial and smooth muscle cells. In this study, we have isolated and functionally characterised a snaclec which we named rhinocetin from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13kDa respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in dose dependent manner, but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP- or thrombin-induced platelet activation. Rhinocetin antagonised the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen induced platelet functions such as fibrinogen binding, calcium mobilisation, granule secretion, aggregation and thrombus formation. It also inhibited integrin α2β1 dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios including haemostasis, thrombosis and envenomation.

Lipoma preferred partner is a mechanosensitive protein regulated by nitric oxide in the heart

Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats (p < 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes (p < 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions (p < 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load.

Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries

Background and Purpose: Calcitonin gene‐related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor‐like receptor (CLR) and receptor activity‐modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR•RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin‐converting enzyme‐1 (ECE‐1). However, it is not known if ECE‐1 regulates the resensitization of CGRP‐induced responses in functional arterial tissue. Experimental Approach: CLR, ECE‐1a‐d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA‐SMCs) and mesenteric arteries was analyzed by RT‐PCR and by immunofluorescence and confocal microscopy. CGRP‐induced signaling in cells was examined by measuring cAMP production and ERK activation. CGRP‐induced relaxation of arteries was measured by isometric wire myography. ECE‐1 was inhibited using the specific inhibitor, SM‐19712. Key Results: RMA‐SMCs and arteries contained mRNA for CLR, ECE‐1a‐d and RAMP1. ECE‐1 was present in early endosomes of RMA‐SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium‐independent relaxation of arteries. ECE‐1 inhibition had no effect on initial CGRP‐induced responses but reduced cAMP generation in RMA‐SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. Conclusions and Implications: ECE‐1 regulates the resensitization of responses to CGRP in RMA‐SMCs and mesenteric arteries. CGRP‐induced relaxation does not involve endothelium‐derived pathways. This is the first report of ECE‐1 regulating CGRP responses in SMCs and arteries. ECE‐1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine.

Possible role for K+ in endothelium-derived hyperpolarizing factor–linked dilatation in rat middle cerebral artery

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF) and K+ are vasodilators in the cerebral circulation. Recently, K+ has been suggested to contribute to EDHF-mediated responses in peripheral vessels. The EDHF response to the protease-activated receptor 2 ligand SLIGRL was characterized in cerebral arteries and used to assess whether K+ contributes as an EDHF. Methods— Rat middle cerebral arteries were mounted in either a wire or pressure myograph. Concentration-response curves to SLIGRL and K+ were constructed in the presence and absence of a variety of blocking agents. In some experiments, changes in tension and smooth muscle cell membrane potential were recorded simultaneously. Results— SLIGRL (0.02 to 20 μmol/L) stimulated concentration and endothelium-dependent relaxation. In the presence of NG-nitro-L-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IKCa, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (1μmol/L), reflecting activation of EDHF. Combined inhibition of KIR with Ba2+ (30μmol/L) and Na+/K+-ATPase with ouabain (1 μmol/L) markedly attenuated the relaxation to EDHF. Raising extracellular [K+] to 15 mmol/L also stimulated smooth muscle relaxation and hyperpolarization, which was also attenuated by combined application of Ba2+ and ouabain. Conclusions— SLIGRL evokes EDHF-mediated relaxation in the rat middle cerebral artery, underpinned by hyperpolarization of the smooth muscle. The profile of blockade of EDHF-mediated hyperpolarization and relaxation supports a pivotal role for IKCa channels. Furthermore, similar inhibition of responses to EDHF and exogenous K+ with Ba2+ and ouabain suggests that K+ may contribute as an EDHF in the middle cerebral artery.

Statins and selective inhibition of Rho kinase protect small conductance calcium-activated potassium channel function (KCa2.3) in cerebral arteries

Background: In rat middle cerebral and mesenteric arteries the KCa2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, KCa2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain KCa2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA). Methods: MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. Results: Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of KCa2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. KCa2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. Conclusions: Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell KCa2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of KCa2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.

Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin.

Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin

T-type Ca2+ channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca2+ channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ∼3 μM) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 μM) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ∼3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation

NK1 receptor stimulation causes contraction and inositol phosphate increase in medium-size human isolated bronchi

Although contraction of human isolated bronchi is mediated mainly by tachykinin NK2 receptors, NK1 receptors, via prostanoid release, contract small-size (approximately 1 mm in diameter) bronchi. Here, we have investigated the presence and biological responses of NK1 receptors in medium-size (2-5 mm in diameter) human isolated bronchi. Specific staining was seen in bronchial sections with an antibody directed against the human NK1 receptor. The selective NK1 receptor agonist, [Sar(9), Met(O2)(11)]SP, contracted about 60% of human isolated bronchial rings. This effect was reduced by two different NK1 receptor antagonists, CP-99,994 and SR 140333. Contraction induced by [Sar(9), Met(O2)(11)]SP was independent of acetylcholine and histamine release and epithelium removal, and was not affected by nitric oxide synthase and cyclooxygenase (COX) inhibition. [Sar(9), Met(O2)(11)]SP increased inositol phosphate (IP) levels, and SR 140333 blocked this increase, in segments of medium- and small-size (approximately 1 mm in diameter) human bronchi. COX inhibition blocked the IP increase induced by [Sar(9), Met(O2)(11)]SP in small-size, but not in medium-size, bronchi. NK1 receptors mediated bronchoconstriction in a large proportion of medium-size human bronchi. Unlike small-size bronchi this effect is independent of prostanoid release, and the results are suggestive of a direct activation of smooth muscle receptors and IP release.

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

Adult craniofacial stem cells: sources and relation to the neural crest

During the process of development, neural crest cells migrate out from their niche between the newly formed ectoderm and the neural tube. Thereafter, they give rise not only to ectodermal cell types, but also to mesodermal cell types. Cell types with neural crest ancestry consequently comprise a number of specialized varieties, such as ectodermal neurons, melanocytes and Schwann cells, as well as mesodermal osteoblasts, adipocytes and smooth muscle cells. Numerous recent studies suggest that stem cells with a neural crest origin persist into adulthood, especially within the mammalian craniofacial compartment. This review discusses the sources of adult neural crest-derived stem cells (NCSCs) derived from the cranium, as well as their differentiation potential and expression of key stem cell markers. Furthermore, the expression of marker genes associated with embryonic stem cells and the issue of multi- versus pluripotency of adult NCSCs is reviewed. Stringent tests are proposed, which, if performed, are anticipated to clarify the issue of adult NCSC potency. Finally, current pre-clinical and clinical data are discussed in light of the clinical impact of adult NCSCs.

Endothelial cell-specific ROS production increases susceptibility to aortic dissection

Background—Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. Methods and Results—A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II–mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45+ inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II–induced vascular smooth muscle cell ROS production. Conclusions—These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II–mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Media damage following detergent sclerotherapy appears to be secondary to the induction of inflammation and apoptosis: an immunohistochemical study elucidating previous histological observations

Objective/Background: Traditionally, sclerotherapy has been thought to work by the cytotoxic effect of the sclerosant upon the endothelium alone. However, studies have shown that sclerotherapy is more successful in smaller veins than in larger veins. This could be explained by the penetration of the sclerosant, or its effect, into the media. This study aimed to investigate intimal and medial damage profiles after sclerosant treatment. Methods: Fresh human varicose veins were treated ex vivo with either 1% or 3% sodium tetradecyl sulphate (STS) for 1 or 10 minutes. The effect of the sclerosant on the vein wall was investigated by immunofluorescent labelling of transverse vein sections using markers for endothelium (CD31), smooth muscle (a-actin), apoptosis (p53) and inflammation (intercellular adhesion molecule-1 [ICAM-1]). Polidocanol (POL; 3%) treatment at 10 minutes was similarly investigated. Results: Endothelial cell death was concentration- and time-dependent for STS but incomplete for both sclerosants. Time, but not concentration, significantly affected cell death (p > .001). A 40% and 30% maximum reduction was observed for STS and POL, respectively. Destruction of 20e30% of smooth muscle cells was found up to 250 mm from the lumen after 3% STS treatment for 10 minutes. POL treatment for 10 minutes showed inferior destruction of medial cells. Following STS treatment and 24-hour tissue culture, p53 and ICAM-1 were upregulated to a depth of around 300 mm. This effect was not observed with POL. Conclusion: Inflammatory and apoptotic markers show the same distribution as medial cell death, implying that sclerotherapy with STS works by inducing apoptosis in the vein wall rather than having an effect restricted to the endothelium. Incomplete loss of endothelial cells and penetration of the sclerosant effect up to 250 mm into the media suggest that medial damage is crucial to the success of sclerotherapy and may explain why it is less effective in larger veins.

Fibroelastic Components of the Vesicourethral Junction of Rats in the Aging Process

The vesicourethral junction comprising the vesical trigone, is relevant in setting and positioning of the urinary bladder, along with the vesical neck, fixed by lateral ligaments of the bladder and tendinous arch of the pelvis fascia. Namely, the puboprostatic ligament (men) and the pubovesical (women). The circular set elastic fibers in this junction are important and valuable in the elasticity and plasticity of the area, allowing quick expansion and withdrawal with the flow of urine, and associated to smooth muscle tissue and nerve control form an important collective to maintain urinary continence. The objective of the present study is to describe the elastic system in the vesicouretral junction in relation to aging and its involvement in the states of urinary continence and incontinence, as well as the study of the vesicouretral junction in various age groups while evaluating with electron transmission microscopy. To carry out the study, 12 Wistar rats were used, divided into groups: neonate (4 animals), adult group (4 animals) and aged group (4 animals). Electron transmission microscopy with use of tanic acid technique associated to glutaraldehyde fixation, satisfactorily showed the extreme structural differences between mature elaunin and oxytalan fibers present between intercelular spaces and bundles of collagen fibers. The phases of elastogenesis in neonate animals and degradation of the elastic system of older animals were also evaluated.

Involvement of the AT(1) receptor in the venoconstriction induced by angiotensin II in both the inferior vena cava and femoral vein

Although angiotensin II-induced venoconstriction has been demonstrated in the rat vena cava and femoral vein, the angiotensin II receptor subtypes (AT(1) or AT(2)) that mediate this phenomenon have not been precisely characterized. Therefore, the present study aimed to characterize the pharmacological receptors involved in the angiotensin II-induced constriction of rat venae cavae and femoral veins, as well as the opposing effects exerted by locally produced prostanoids and NO upon induction of these vasomotor responses. The obtained results suggest that both AT(1) and AT(2) angiotensin II receptors are expressed in both veins. Angiotensin II concentration-response curves were shifted toward the right by losartan but not by PD 123319 in both the vena cava and femoral vein. Moreover, it was observed that both 10(-5) M indomethacin and 10(-4) M L-NAME improve the angiotensin II responses in the vena cava and femoral vein. In conclusion, in the rat vena cava and femoral vein, angiotensin II stimulates AT(1) but not AT(2) to induce venoconstriction, which is blunted by vasodilator prostanoids and NO. (C) 2010 Elsevier Inc. All rights reserved.

Acute actions of thyroid hormone on blood vessel biochemistry and physiology

Purpose of review Description of the progress about the vascular effects promoted by thyroid hormones. Recent findings Over the past few years, a number of studies have shown that in addition to genomic effects on blood vessels, thyroid hormones exert extranuclear nongenomic effects on vascular smooth muscle cells and endothelium. These nongenomic effects occur rapidly and do not involve thyroid hormone response elements-mediated transcriptional events. In this context, the genomic and nongenomic events promoted by thyroid hormones act in concert to control the vascular hemodynamic and regulate the cardiovascular function. Summary Considering the antiatherogenic property of thyroid hormones and the rapid effects produced by this molecule as a vasodilator, including that in the coronary bed, a better understanding of the molecular mechanisms involved in its action may contribute to the development of drugs that can be clinically used to increase the known benefits promoted by thyroid hormones in cardiovascular physiology.