Tissue-specific early and late activated lymphocytes immunophenotype in chronic rhinosinusitis with nasal polyps.

KEY POINTS: T-cell activation in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) is enriched by late cytotoxic T cells. The proportion of early and intermediate activated cytotoxic T cells decreases in nasal polyps of patients with CRSwNP. Our results identify late activated cytotoxic T cells as potential biomarkers or therapeutic targets for patients with CRSwNP.

Role of Fibroblasts in Chronic Inflammatory Signalling in Chronic Rhinosinusitis with Nasal Polyps-A Systematic Review.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory disease of the nose and paranasal sinuses characterized by the presence of nasal polyps. The symptoms produced by the presence of nasal polyps such as nasal obstruction, nasal discharge, facial pain, headache, and loss of smell cause a worsening in the quality of life of patients. The source of the nasal polyps remains unclear, although it seems to be due to a chronic inflammation process in the sinonasal mucosa. Fibroblasts, the main cells in connective tissue, are intimately involved in the inflammation processes of various diseases; to this end, we carried out a systematic review to evaluate their inflammatory role in nasal polyps. Thus, we evaluated the main cytokines produced by nasal polyp-derived fibroblasts (NPDF) to assess their involvement in the production of nasal polyps and their involvement in different inflammatory pathways. The results of the review highlight the inflammatory role of NPDF through the secretion of various cytokines involved in the T1, T2, and T3 inflammatory pathways, as well as the ability of NPDF to be stimulated by a multitude of substances. With these findings, the fibroblast is positioned as a new potential therapeutic target in the treatment of CRSwNP.

PKCα-Mediated Downregulation of RhoA Activity in Depolarized Vascular Smooth Muscle: Synergistic Vasorelaxant Effect of PKCα and ROCK Inhibition.

BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. METHODS: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. RESULTS: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. CONCLUSION: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.

Contribution of L-type Ca2+ channel-sarcoplasmic reticulum coupling to depolarization-induced arterial contraction in spontaneously hypertensive rats.

Evidence has shown that vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) are depolarized and that the expression of L-type Ca2+ channels (LTCCs) and the sarcoplasmic reticulum (SR) Ca2+ buffering system are upregulated. Arterial rings exposed to high K+ solutions develop a contraction with two components, namely, an initial or phasic component and a sustained or tonic component. Because LTCCs and SR have different functions in the phasic and tonic components of depolarization-induced contraction, this study investigated the role of LTCC-SR coupling in depolarized arterial rings of SHRs. In the absence of extracellular Ca2+, high external K+ or LTCC agonists elicited a transitory contraction, which was sensitive to nifedipine and was potentiated in SHRs. In the presence of extracellular Ca2+, cyclopiazonic acid (CPA), an SR Ca2+-ATPase (SERCA) inhibitor, evoked a transient contraction that was significantly increased in SHRs. Although the phasic and tonic components were markedly increased in depolarized arterial rings of SHRs, they showed different voltage-dependence and sensitivity to SERCA inhibition. The tonic component was more sensitive to moderate depolarizations, and CPA selectively reduced the tonic component to the level observed in WKY rats. These results suggested that LTCC-SR coupling is potentiated in the sustained contraction of hypertensive VSMCs.

Regulation of RhoA/ROCK and sustained arterial contraction by low cytosolic Ca2+ levels during prolonged depolarization of arterial smooth muscle.

The role of L-type Ca2+ channels (LTCCs) and RhoA/Rho kinase (ROCK) on depolarization-induced sustained arterial contraction lasting several minutes is already known. However, in vivo, vascular smooth muscle cells can be depolarized for longer periods, inducing substantial inactivation of LTCCs and markedly reducing Ca2+ influx into the myocytes. We have examined, in femoral arterial rings, the role of LTCCs and RhoA/ROCK during long-lasting depolarization. Our results reveal a new vasoreactive response after 20-30min of depolarization in 2.5mM external Ca2+ that has not been identified previously with shorter stimuli. Prolonged depolarization-induced arterial contraction was permanently abolished when arterial rings were treated with 100nM external Ca2+ or 20nM nifedipine. However, when Ca2+ influx was restricted, applying ~7µM external Ca2+ solution or 3nM nifedipine, vasorelaxation was transient, and isometric force slowly increased after 30min and maintained its level until the end of the stimulus. Under these conditions, arterial contraction showed the same temporal course of RhoA activity and was sensitive to fasudil, nifedipine and cyclopiazonic acid. Ca2+-response curve in ß-escin permeabilized arteries was also sensitive to ROCK inhibitors. Thus, although long-lasting depolarization inactivates LTCCs, the reduced Ca2+ entry can induce a detectable arterial contraction via RhoA/ROCK activation.

Role of L-type Ca(2+) channels, sarcoplasmic reticulum and Rho kinase in rat basilar artery contractile properties in a new model of subarachnoid hemorrhage.

We have previously described that L-type Ca(2+) channels' (LTCCs) activation and metabotropic Ca(2+) release from the sarcoplasmic reticulum (SR) regulate RhoA/Rho kinase (ROCK) activity and sustained arterial contraction. We have investigated whether this signaling pathway can be altered in a new experimental model of subarachnoid hemorrhage (SAH). For this purpose, arterial reactivity was evaluated on days 1 to 5 after surgery. A significant increase of basal tone, measured 4 and 60min after normalization, was observed on day 5 after SAH and at 60min on days 2 and 3 after SAH. This phenomenon was suppressed with LTCCs and ROCK inhibitors. We have also studied arterial rings vasoreactivity in response to high K(+) solutions. Interestingly, there were no significant differences in the phasic component of the high K(+)-induced contraction between sham and SAH groups, whereas a significant increase in the sustained contraction was observed on day 5 after SAH. This latter component was sensitive to fasudil, and selectively reduced by low nifedipine concentration, and phospholipase C and SR-ATPase inhibitors. Therefore, our data suggest that the metabotropic function of LTCCs is potentiated in SAH. Our results could provide a new strategy to optimize the pharmacological treatment of this pathological process.

Low-dose combination of Rho kinase and L-type Ca(2+) channel antagonists for selective inhibition of depolarization-induced sustained arterial contraction.

L-type Ca(2+) channels (LTCCs) are involved in the maintenance of tonic arterial contractions and regulate the RhoA/Rho-associated kinase (ROCK) sensitization cascade. We have tested effects of individual and combined low concentrations of LTCCs and ROCK inhibitors to produce arterial relaxation without the adverse side effects of LTCCs antagonists. We have also studied whether this pharmacological strategy alters Ca(2+)-dependent electrical properties of isolated arterial and cardiac myocytes as well as cardiac contractility. Rat basilar, human carotid and coronary arterial rings were mounted on a small-vessel myograph to measure isometric tension and cardiac contractility was measured in Langendorff-perfused rat heart. Simultaneous cytosolic Ca(2+) concentration and arterial diameter were measured in intact pressurized arteries loaded with Fura-2. Patch-clamp techniques were used to measure electrical properties in isolated cardiac and arterial myocytes. Low concentrations of LTCCs and ROCK inhibitors reduced the tonic component of moderate depolarization-evoked contraction, leaving the phasic component practically unaltered. This selective vasorelaxant effect was more marked when the LTCCs and ROCK inhibitors were applied together. In the concentration range used (nM), Ca(2+) currents in arterial myocytes, cardiac action potentials and heart contractility were unaffected by this pharmacological approach. In conclusion, low doses of LTCCs and ROCK inhibitors could be used to selectively relax precontracted arteries in pathologic conditions such as hypertension, and cerebral or coronary spasms with minor side effects on physiological contractile properties of vascular and cardiac myocytes.

Contractile responses to rat urotensin II in resting and depolarized basilar arteries

The effects of human urotensin II (hUII) on the vascular tone of different animal species has been studied extensively. However, little has been reported on the vasoactive effects of rat urotensin (rUII) in murine models. The aim of the present study was to investigate the effects of rUII on vasoreactivity in rat basilar arteries. Basilar arteries from adult male Wistar rats (300-350 g) were isolated, cut in rings, and mounted on a small vessel myograph to measure isometric tension. rUII concentrations were studied in both resting and depolarized state. To remove endothelial nitric oxide effects from the rUII response, we treated selected arterial rings with Nω-nitro-L-arginine methyl ester (L-NAME). 10 μM rUII produced a potent vasoconstrictor response in rat basilar arteries with intact endothelium, while isometric forces remained unaffected in arterial rings treated with lower rUII concentrations. Although L-NAME did not have a significant effect on 10 μM rUII-evoked contraction, it slightly increased arterial ring contraction elicited by 1 μM rUII. In depolarized arteries, dose-dependent rUII increased depolarization-induced contractions. This effect was suppressed by L-NAME. Our results show that the rat basilar artery has a vasoconstrictor response to rUII. The most potent vasoconstrictor effect was produced by lower doses of rUII (0.1 and 1 μM) in depolarized arteries with intact endothelium. This effect could facilitate arterial vasospasm in vascular pathophysiological processes such as subarachnoid hemorrhage and hypertension, when sustained depolarization and L-type Ca2+ channel activation are present

Contractile responses to rat urotensin II in resting and depolarized basilar arteries.

The effects of human urotensin II (hUII) on the vascular tone of different animal species has been studied extensively. However, little has been reported on the vasoactive effects of rat urotensin (rUII) in murine models. The aim of the present study was to investigate the effects of rUII on vasoreactivity in rat basilar arteries. Basilar arteries from adult male Wistar rats (300-350 g) were isolated, cut in rings, and mounted on a small vessel myograph to measure isometric tension. rUII concentrations were studied in both resting and depolarized state. To remove endothelial nitric oxide effects from the rUII response, we treated selected arterial rings with Nω-nitro-L-arginine methyl ester (L-NAME). 10 µM rUII produced a potent vasoconstrictor response in rat basilar arteries with intact endothelium, while isometric forces remained unaffected in arterial rings treated with lower rUII concentrations. Although L-NAME did not have a significant effect on 10 µM rUII-evoked contraction, it slightly increased arterial ring contraction elicited by 1 µM rUII. In depolarized arteries, dose-dependent rUII increased depolarization-induced contractions. This effect was suppressed by L-NAME. Our results show that the rat basilar artery has a vasoconstrictor response to rUII. The most potent vasoconstrictor effect was produced by lower doses of rUII (0.1 and 1 µM) in depolarized arteries with intact endothelium. This effect could facilitate arterial vasospasm in vascular pathophysiological processes such as subarachnoid hemorrhage and hypertension, when sustained depolarization and L-type Ca(2+) channel activation are present.

A new metabotropic role for L-type Ca(2+) channels in vascular smooth muscle contraction.

Vascular smooth muscle cells (VSMCs) contraction can be evoked by the rise of cytosolic [Ca(2+)] owing to transmembrane Ca(2+) influx or sarcoplasmic reticulum (SR) Ca(2+) release. Although the classical ionotropic role of voltagedependent (L-type) Ca(2+) channels (VGCCs) is known, we review here data suggesting a new metabotropic function of VGCCs in vascular smooth muscle cells. VGCCs can trigger Ca(2+) release from the SR in the absence of extracellular Ca2+. During depolarization, VGCCs can activate G proteins and phospholipase C (PLC)/inositol 1,4,5-trisphosphate (InsP3) pathway leading to Ca2+ release and arterial contraction. This new metabotropic role of VGCCs, referred as calcium channel-induced Ca(2+) release (CCICR), has a major role in tonic VSM contractility, as it links sustained membrane depolarization and Ca(2+) channel activation with metabotropic Ca(2+) release from the sarcoplasmic reticulum (SR) and tonic smooth muscle contraction. This new role of VGCCs could have a wide functional relevance for the pathogenesis of vasospasms mediated by membrane depolarization and vasoactive agents that can activate VGCCs. Precise understanding of CCICR could help to optimize pharmacological treatments for clinical conditions where Ca(2+) channels antagonists are recommended.

Tonic arterial contraction mediated by L-type Ca2+ channels requires sustained Ca2+ influx, G protein-associated Ca2+ release, and RhoA/ROCK activation.

KCl-evoked sustained contraction requires L-type Ca(2+) channel activation, metabotropic Ca(2+) release from the sarcoplasmic reticulum (mechanism denoted calcium channel-induced Ca(2+) release) and RhoA/Rho associated kinase activation. Although high K(+) solutions are used to depolarize myocytes, these solutions can stimulate other signaling pathways such as those triggered by the activation of muscarinic and purinergic receptors. The present study examines the functional role of calcium channel-induced Ca(2+) release under pharmacological activation of L-type Ca(2+) channel without significant membrane depolarization. It also analyzes the role of the "steady-state" Ca(2+) influx through L-type Ca(2+) channels on myocyte sustained contraction. Measurement of contractility in arterial rings was done on a vessel myograph. Membrane potential was measured by fluorescence techniques loading intact myocytes with a membrane potential sensitive dye, and a reversible permeabilization method was used to load myocytes in intact arteries with GDPßS and Ca(v)1.2 siRNA. Application of an L-type Ca(2+) channel agonist, without effect on membrane potential, evoked sustained contraction via G-protein induced Ca(2+) release from the sarcoplasmic reticulum and RhoA/Rho associated kinase activation. Tonic myocyte contractions mediated by L-type Ca(2+) channel activation required sustained Ca(2+) influx through the channels and Ca(2+) uptake by the sarcoplasmic reticulum. Because L-type Ca(2+) channels participate in numerous pathophysiological processes mediated by maintained arterial contraction, our data could help to optimize therapeutic treatment of arterial vasospasm.

Tungstate activates BK channels in a ß subunit- and Mg2+-dependent manner: relevance for arterial vasodilatation.

AIMS: Tungstate reduces blood pressure in experimental animal models of both hypertension and metabolic syndrome, although the underlying mechanisms are not fully understood. Given that the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel is a key element in the control of arterial tone, our aim was to evaluate whether BK channel modulation by tungstate can contribute to its antihypertensive effect. METHODS AND RESULTS: Patch-clamp studies of heterologously expressed human BK channels (α + ß(1-4) subunits) revealed that cytosolic tungstate (1 mM) induced a significant left shift (∼20 mV) in the voltage-dependent activation curve only in BK channels containing αß(1) or αß(4) subunits, but reduced the amplitude of K(+) currents through all BK channels tested. The ß(1)-dependent activation of BK channels by tungstate was enhanced at cytosolic Ca(2+) levels reached during myocyte contraction, and prevented either by removal of cytosolic Mg(2+) or by mutations rendering the channel insensitive to Mg(2+). A lower concentration of tungstate (0.1 mM) induced voltage-dependent activation of the vascular BKαß(1) channel without reducing current amplitude, and consistently exerted a vasodilatory action on wild-type but not on ß(1)-knockout mouse arteries pre-contracted with endothelin-1. CONCLUSION: Tungstate activates BK channels in a ß subunit- and Mg(2+)-dependent manner and induces vasodilatation only in mouse arteries that express the BK ß(1) subunit.

Metabotropic regulation of RhoA/Rho-associated kinase by L-type Ca2+ channels: new mechanism for depolarization-evoked mammalian arterial contraction.

BACKGROUND: Sustained vascular smooth muscle contraction is mediated by extracellular Ca(2+) influx through L-type voltage-gated Ca(2+) channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca(2+) sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca(2+) release from the sarcoplasmic reticulum (calcium channel-induced Ca(2+) release). We have studied the functional role of calcium channel-induced Ca(2+) release and the inter-relations between Ca(2+) channel and RhoA/ROCK activation. METHODS AND RESULTS: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca(2+) and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca(2+) channel activation and subsequent metabotropic Ca(2+) release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca(2+) from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca(2+) release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca(2+) influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. CONCLUSIONS: These findings reveal that calcium channel-induced Ca(2+) release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca(2+) channel activation with metabotropic Ca(2+) release and sensitization (RhoA/ROCK stimulation).