Tachycardia evoked from insular stroke in rats is dependent on glutamatergic neurotransmission in the dorsomedial hypothalamus.

BACKGROUND AND PURPOSE: Damage to the insula results in cardiovascular complications. In rats, activation of N-methyl-d-aspartate receptors (NMDARs) in the intermediate region of the posterior insular cortex (iIC) results in sympathoexcitation, tachycardia and arterial pressure increases. Similarly, focal experimental hemorrhage at the iIC results in a marked sympathetic-mediated increase in baseline heart rate. The dorsomedial hypothalamic region (DMH) is critical for the integration of sympathetic-mediated tachycardic responses. Here, whether responses evoked from the iIC are dependent on a synaptic relay in the DMH was evaluated. METHODS: Wistar rats were prepared for injections into the iIC and DMH. Anatomical (tracing combined with immunofluorescence) and functional experiments (cardiovascular and sympathetic recordings) were performed. RESULTS: The iIC sends dense projections to the DMH. Approximately 50% of iIC neurons projecting to the DMH express NMDARs, NR1 subunit. Blockade of glutamatergic receptors in the DMH abolishes the cardiovascular and autonomic responses evoked by the activation of NMDARs in the iIC (change in mean arterial pressure 7 ± 1 vs. 1 ± 1 mmHg after DMH blockade; change in heart rate 28 ± 3 vs. 0 ± 3 bpm after DMH blockade; change in renal sympathetic nerve activity 23% ± 1% vs. -1% ± 4% after DMH blockade). Experimental hemorrhage at the iIC resulted in a marked tachycardia (change 89 ± 14 bpm) that was attenuated by 65% ± 5% (p = 0.0009) after glutamatergic blockade at the DMH. CONCLUSIONS: The iIC-induced tachycardia is largely dependent upon a glutamatergic relay in the DMH. Our study reveals the presence of an excitatory glutamatergic pathway from the iIC to the DMH that may be involved in the cardiovascular alterations observed after insular stroke.

Interleukin-6 trans-signaling contributes to chronic hypoxia-induced pulmonary hypertension.

Interleukin-6 (IL-6) is a pleotropic cytokine that signals through the membrane-bound IL-6 receptor (mIL-6R) to induce anti-inflammatory ("classic-signaling") responses. This cytokine also binds to the soluble IL-6R (sIL-6R) to promote inflammation ("trans-signaling"). mIL-6R expression is restricted to hepatocytes and immune cells. Activated T cells release sIL-6R into adjacent tissues to induce trans-signaling. These cellular actions require the ubiquitously expressed membrane receptor gp130. Reports show that IL-6 is produced by pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia in culture as well as the medial layer of the pulmonary arteries in mice exposed to chronic hypoxia (CH), and IL-6 knockout mice are protected from CH-induced pulmonary hypertension (PH). IL-6 has the potential to contribute to a broad array of downstream effects, such as cell growth and migration. CH-induced PH is associated with increased proliferation and migration of PASMCs to previously non-muscularized vessels of the lung. We tested the hypothesis that IL-6 trans-signaling contributes to CH-induced PH and arterial remodeling. Plasma levels of sgp130 were significantly decreased in mice exposed to CH (380 mmHg) for five days compared to normoxic control mice (630 mmHg), while sIL-6R levels were unchanged. Consistent with our hypothesis, mice that received the IL-6 trans-signaling-specific inhibitor sgp130Fc, a fusion protein of the soluble extracellular portion of gp130 with the constant portion of the mouse IgG1 antibody, showed attenuation of CH-induced increases in right ventricular systolic pressure, right ventricular and pulmonary arterial remodeling as compared to vehicle (saline)-treated control mice. In addition, PASMCs cultured in the presence of IL-6 and sIL-6R showed enhanced migration but not proliferation compared to those treated with IL-6 or sIL-6R alone or in the presence of sgp130Fc. These results indicate that IL-6 trans-signaling contributes to pulmonary arterial cell migration and CH-induced PH.

Endothelial CaMKII as a regulator of eNOS activity and NO-mediated vasoreactivity.

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase important in transducing intracellular Ca2+ signals. While in vitro data regarding the role of CaMKII in the regulation of endothelial nitric oxide synthase (eNOS) are contradictory, its role in endothelial function in vivo remains unknown. Using two novel transgenic models to express CaMKII inhibitor peptides selectively in endothelium, we examined the effect of CaMKII on eNOS activation, NO production, vasomotor tone and blood pressure. Under baseline conditions, CaMKII activation was low in the aortic wall. Consistently, systolic and diastolic blood pressure, heart rate and plasma NO levels were unaltered by endothelial CaMKII inhibition. Moreover, endothelial CaMKII inhibition had no significant effect on NO-dependent vasodilation. These results were confirmed in studies of aortic rings transduced with adenovirus expressing a CaMKII inhibitor peptide. In cultured endothelial cells, bradykinin treatment produced the anticipated rapid influx of Ca2+ and transient CaMKII and eNOS activation, whereas CaMKII inhibition blocked eNOS phosphorylation on Ser-1179 and dephosphorylation at Thr-497. Ca2+/CaM binding to eNOS and resultant NO production in vitro were decreased under CaMKII inhibition. Our results demonstrate that CaMKII plays an important role in transient bradykinin-driven eNOS activation in vitro, but does not regulate NO production, vasorelaxation or blood pressure in vivo under baseline conditions.

NFATc3 and VIP in Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease.

Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are both debilitating lung diseases which can lead to hypoxemia and pulmonary hypertension (PH). Nuclear Factor of Activated T-cells (NFAT) is a transcription factor implicated in the etiology of vascular remodeling in hypoxic PH. We have previously shown that mice lacking the ability to generate Vasoactive Intestinal Peptide (VIP) develop spontaneous PH, pulmonary arterial remodeling and lung inflammation. Inhibition of NFAT attenuated PH in these mice suggesting a connection between NFAT and VIP. To test the hypotheses that: 1) VIP inhibits NFAT isoform c3 (NFATc3) activity in pulmonary vascular smooth muscle cells; 2) lung NFATc3 activation is associated with disease severity in IPF and COPD patients, and 3) VIP and NFATc3 expression correlate in lung tissue from IPF and COPD patients. NFAT activity was determined in isolated pulmonary arteries from NFAT-luciferase reporter mice. The % of nuclei with NFAT nuclear accumulation was determined in primary human pulmonary artery smooth muscle cell (PASMC) cultures; in lung airway epithelia and smooth muscle and pulmonary endothelia and smooth muscle from IPF and COPD patients; and in PASMC from mouse lung sections by fluorescence microscopy. Both NFAT and VIP mRNA levels were measured in lungs from IPF and COPD patients. Empirical strategies applied to test hypotheses regarding VIP, NFATc3 expression and activity, and disease type and severity. This study shows a significant negative correlation between NFAT isoform c3 protein expression levels in PASMC, activity of NFATc3 in pulmonary endothelial cells, expression and activity of NFATc3 in bronchial epithelial cells and lung function in IPF patients, supporting the concept that NFATc3 is activated in the early stages of IPF. We further show that there is a significant positive correlation between NFATc3 mRNA expression and VIP RNA expression only in lungs from IPF patients. In addition, we found that VIP inhibits NFAT nuclear translocation in primary human pulmonary artery smooth muscle cells (PASMC). Early activation of NFATc3 in IPF patients may contribute to disease progression and the increase in VIP expression could be a protective compensatory mechanism.

Vasculo-Neuronal Coupling: Retrograde Vascular Communication to Brain Neurons.

Continuous cerebral blood flow is essential for neuronal survival, but whether vascular tone influences resting neuronal function is not known. Using a multidisciplinary approach in both rat and mice brain slices, we determined whether flow/pressure-evoked increases or decreases in parenchymal arteriole vascular tone, which result in arteriole constriction and dilation, respectively, altered resting cortical pyramidal neuron activity. We present evidence for intercellular communication in the brain involving a flow of information from vessel to astrocyte to neuron, a direction opposite to that of classic neurovascular coupling and referred to here as vasculo-neuronal coupling (VNC). Flow/pressure increases within parenchymal arterioles increased vascular tone and simultaneously decreased resting pyramidal neuron firing activity. On the other hand, flow/pressure decreases evoke parenchymal arteriole dilation and increased resting pyramidal neuron firing activity. In GLAST-CreERT2; R26-lsl-GCaMP3 mice, we demonstrate that increased parenchymal arteriole tone significantly increased intracellular calcium in perivascular astrocyte processes, the onset of astrocyte calcium changes preceded the inhibition of cortical pyramidal neuronal firing activity. During increases in parenchymal arteriole tone, the pyramidal neuron response was unaffected by blockers of nitric oxide, GABAA, glutamate, or ecto-ATPase. However, VNC was abrogated by TRPV4 channel, GABAB, as well as an adenosine A1 receptor blocker. Differently to pyramidal neuron responses, increases in flow/pressure within parenchymal arterioles increased the firing activity of a subtype of interneuron. Together, these data suggest that VNC is a complex constitutive active process that enables neurons to efficiently adjust their resting activity according to brain perfusion levels, thus safeguarding cellular homeostasis by preventing mismatches between energy supply and demand. SIGNIFICANCE STATEMENT: We present evidence for vessel-to-neuron communication in the brain slice defined here as vasculo-neuronal coupling. We showed that, in response to increases in parenchymal arteriole tone, astrocyte intracellular Ca2+ increased and cortical neuronal activity decreased. On the other hand, decreasing parenchymal arteriole tone increased resting cortical pyramidal neuron activity. Vasculo-neuronal coupling was partly mediated by TRPV4 channels as genetic ablation, or pharmacological blockade impaired increased flow/pressure-evoked neuronal inhibition. Increased flow/pressure-evoked neuronal inhibition was blocked in the presence of adenosine A1 receptor and GABAB receptor blockade. Results provide evidence for the concept of vasculo-neuronal coupling and highlight the importance of understanding the interplay between basal CBF and resting neuronal activity.

Mechanisms of NFATc3 activation by increased superoxide and reduced hydrogen peroxide in pulmonary arterial smooth muscle.

We recently demonstrated increased superoxide (O2(·-)) and decreased H2O2 levels in pulmonary arteries of chronic hypoxia-exposed wild-type and normoxic superoxide dismutase 1 (SOD1) knockout mice. We also showed that this reciprocal change in O2(·-) and H2O2 is associated with elevated activity of nuclear factor of activated T cells isoform c3 (NFATc3) in pulmonary arterial smooth muscle cells (PASMC). This suggests that an imbalance in reactive oxygen species levels is required for NFATc3 activation. However, how such imbalance activates NFATc3 is unknown. This study evaluated the importance of O2(·-) and H2O2 in the regulation of NFATc3 activity. We tested the hypothesis that an increase in O2(·-) enhances actin cytoskeleton dynamics and a decrease in H2O2 enhances intracellular Ca(2+) concentration, contributing to NFATc3 nuclear import and activation in PASMC. We demonstrate that, in PASMC, endothelin-1 increases O2(·-) while decreasing H2O2 production through the decrease in SOD1 activity without affecting SOD protein levels. We further demonstrate that O2(·-) promotes, while H2O2 inhibits, NFATc3 activation in PASMC. Additionally, increased O2(·-)-to-H2O2 ratio activates NFATc3, even in the absence of a Gq protein-coupled receptor agonist. Furthermore, O2(·-)-dependent actin polymerization and low intracellular H2O2 concentration-dependent increases in intracellular Ca(2+) concentration contribute to NFATc3 activation. Together, these studies define important and novel regulatory mechanisms of NFATc3 activation in PASMC by reactive oxygen species.

NFAT is required for spontaneous pulmonary hypertension in superoxide dismutase 1 knockout mice.

Elevated reactive oxygen species are implicated in pulmonary hypertension (PH). Superoxide dismutase (SOD) limits superoxide bioavailability, and decreased SOD activity is associated with PH. A decrease in SOD activity is expected to increase superoxide and reduce hydrogen peroxide levels. Such an imbalance of superoxide/hydrogen peroxide has been implicated as a mediator of nuclear factor of activated T cells (NFAT) activation in epidermal cells. We have shown that NFATc3 is required for chronic hypoxia-induced PH. However, it is unknown whether NFATc3 is activated in the pulmonary circulation in a mouse model of decreased SOD1 activity and whether this leads to PH. Therefore, we hypothesized that an elevated pulmonary arterial superoxide/hydrogen peroxide ratio activates NFATc3, leading to PH. We found that SOD1 knockout (KO) mice have elevated pulmonary arterial wall superoxide and decreased hydrogen peroxide levels compared with wild-type (WT) littermates. Right ventricular systolic pressure (RVSP) was elevated in SOD1 KO and was associated with pulmonary arterial remodeling. Vasoreactivity to endothelin-1 was also greater in SOD1 KO vs. WT mice. NFAT activity and NFATc3 nuclear localization were increased in pulmonary arteries from SOD1 KO vs. WT mice. Administration of A-285222 (selective NFAT inhibitor) decreased RVSP, arterial wall thickness, vasoreactivity, and NFAT activity in SOD1 KO mice to WT levels. The SOD mimetic, tempol, also reduced NFAT activity, NFATc3 nuclear localization, and RVSP to WT levels. These findings suggest that an elevated superoxide/hydrogen peroxide ratio activates NFAT in pulmonary arteries, which induces vascular remodeling and increases vascular reactivity leading to PH.

Molecular weight of different angiotensin II polymers directly determines: density of endothelial membrane AT1 receptors and coronary vasoconstriction.

We have shown that angiotensin II (Ang II) does not diffuse across the vessel wall, remaining intravascularly confined and acting solely on the coronary endothelial luminal membrane (CELM) receptors. A sustained intracoronary infusion of Ang II causes transient coronary vasoconstriction (desensitization) due to membrane internalization of CELM Ang II type 1 receptors (CELM-AT1R). In contrast, sustained intracoronary infusion of a non-diffusible polymer of Ang II (Ang II-Pol, 15,000 kDa) causes a sustained vasoconstriction by preventing CELM-AT1R internalization. In addition, a sustained intracoronary infusion of Ang II leads to a depressed response following a secondary Ang II administration (tachyphylaxis) that is reversed by Ang II-Pol. These findings led us to hypothesize that the rate of desensitization, tachyphylaxis, and AT1R internalization were dependent on Ang II-Pol molecular weight. To test this hypothesis, we synthesized Ang II-Pols of the following molecular weights (in kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15,000. Vasoconstriction was measured following intracoronary infusion of Ang II-Pols in Langendorff-perfused guinea pig hearts at constant flow. The CELM protein fraction was extracted using the silica pellicle technique at different time points in order to determine the rate of AT1R internalization following each Ang II-Pol infusion. CELM-AT1R density was quantified by Western blot. We found that the rate of desensitization and the tachyphylaxis effect varied inversely with the molecular weight of the Ang II-Pols. Inversely proportional to the molecular weight of Ang II-Pol the CELM-AT1R density decreases over time. These results indicate that the mechanism responsible for the decreased rate of desensitization and tachyphylaxis by higher molecular weight Ang II polymers is due to reduction in the rate of CELM-AT1R internalization. These Ang II polymers would be valuable tools for studying the relationship between AT1R internalization and physiological effects.

The coronary endothelium behaves as a functional diffusion barrier for intravascular Angiotensin II.

Diverse intracoronary hormones cause their cardiac effects solely via activation of their coronary endothelial luminal membrane (CELM) receptors. To test this hypothesis for Ang II, we synthesized: a) two large polymers of Ang II (Ang II-POL) and Losartan (Los-POL) which act only in the CELM's AT1R because they cannot cross the endothelial barrier and b) biotin-labeled Ang II (Ang II-Biotin) and Ang II-POL-Biotin to be identified by microscopy in tissues. Sustained coronary perfusion of Ang II (potentially diffusible) or Ang II-POL caused a positive inotropic effect (PIE) and an increase in coronary perfusion pressure (CPP) of equal magnitude that were blocked by Losartan and Los-POL. However, Ang II effects, in contrast to Ang II-POL effects, were transient due to desensitization and resulted in tachyphylaxis to a second administration of Ang II or Ang II-POL. Furthermore, if Ang II and Ang II-POL acted differently on the same receptor; a competition of effects would be expected. This was demonstrated by infusing simultaneously a molar ratio of Ang II:Ang II-POL. As this molar ratio decreased, Ang II-induced desensitization and tachyphylaxis decreased. Intravascularly-administered Ang II-Biotin and Ang II-POL-Biotin remained bound and confined to the endothelium. Our results support the hypothesis and indicate intravascular Ang II, not by mass exchange with the interstitium, but by an action restricted to the CELM's AT1R, causes release of endothelial chemical messengers that exert physiological effects and modulate the effects and metabolism of paracrine Ang II. Endocrine Ang II controls and communicates with its paracrine counterparts solely through endothelial receptors.

Luminal endothelial lectins with affinity for N-acetylglucosamine determine flow-induced cardiac and vascular paracrine-dependent responses.

Coronary blood flow applied to the endothelial lumen modulates parenchymal functions via paracrine effectors, but the mechanism of flow sensation is unknown. We and others have demonstrated that coronary endothelial luminal membrane (CELM) oligosaccharides and lectins are involved in flow detection, and we proposed that cardiac effects of coronary flow result from a reversible flow-modulated lectin-oligosaccharide interaction. Recently, glycosylated and amiloride-sensitive Na(+)/Ca(++) channels (ENaCs) have been proposed to be involved in the flow-induced endothelial responses. Because N-acetylglucosamine (GlcNac) is one of the main components of glycocalyx oligosaccharides (i.e., hyaluronan [-4GlcUAbeta1-3GlcNAcbeta1-](n)), the aim of this article is to isolate and define CELM GlcNac-binding lectins and determine their role in cardiac and vascular flow-induced effects. For this purpose, we synthesized a 460-kDa GlcNac polymer (GlcNac-Pol) with high affinity toward GlcNac-recognizing lectins. In the heart, intracoronary administration of GlcNac-Pol upon binding to CELM diminishes the flow-dependent positive inotropic and dromotropic effects. Furthermore, GlcNac-Pol was used as an affinity probe to isolate CELM GlcNac-Pol-recognizing lectins and at least 35 individual lectinic peptides were identified, one of them the beta-ENaC channel. Some of these lectins could participate in flow sensing and in GlcNac-Pol-induced effects. We also adopted a flow-responsive and well-accepted model of endothelial-parenchymal paracrine interaction: isolated blood vessels perfused at controlled flow rates. We established that flow-induced vasodilatation (FIV) is blocked by endothelial luminal membrane (ELM) bound GlcNac-Pol, nitro-l-arginine methyl ester and indomethacin, amiloride, and hyaluronidase. The effect of hyaluronidase was reversed by infusion of soluble hyaluronan. These results indicate that GlcNac-Pol inhibits FIV by competing and displacing intrinsic hyaluronan bound to a lectinic structure such as the amiloride-sensitive ENaC. Nitric oxide and prostaglandins are the putative paracrine mediators of FIV.

Two dissimilar AT(1) agonists distinctively activate AT(1) receptors located on the luminal membrane of coronary endothelium.

Diverse intracoronary agonists cause cardiac effects while acting on coronary endothelial luminal membrane (CELM) receptor. Our data show: a) the presence of AT(1)R in isolated CELM and in all cardiac cell types and b) sustained intracoronary infusions of Ang II-POL, a large sized molecule (approximately 15,000 kDa) confined to the vessel lumen that can only act on CELM's AT(1)R or Ang II (approximately 1 kDa); both exert the same maximum positive inotropic (PIE) and coronary constriction (CPP). The effects of these two agonists are blocked by Losartan and by Sar-POL; a large size antagonist (approximately 15,000 kDa) that acts only on CELM. Ang II effects are transient due to desensitization and cause tachyphylaxis to Ang II and toward Ang II-POL suggesting that both Ang II and Ang II-POL act on the same receptor group. In contrast, Ang II-POL effects are sustained and do not cause tachyphylaxis. The results show that intravascular Ang II and Ang II-POL act differentially by an unknown mechanism on CELM's AT(1)R and suggest that intravascular Ang II and Ang II-POL cause PIE and CCP by activation limited to CELM's AT(1)R through an unknown mechanism that is space-confined to the CELM's AT(1)R.

Involvement of endothelial Man and Gal-binding lectins in sensing the flow in coronary arteries.

The coronary endothelial luminal membrane (CELM) glycocalyx has diverse molecules involved in blood flow signal transduction. Evidence suggests that some of these structures may be lectinic. To test this, we synthesized two monosaccharide polymers (Mon-Pols) made of Mannose (Man-Pol) or Galactose (Gal-Pol) covalently coupled to Dextran (70 kDa) and used them as lectin affinity probes. In situ intracoronary infusion of both polymers resulted in CELM-binding but only Man-Pol caused a reduction in flow-induced positive inotropism and dromotropism. To demonstrate that our lectinic probes could bind to CELM lectins, a representative CELM protein fraction was isolated via intracoronary infusion of a cationic silica colloid and either Mannose- or Galactose-binding lectins were purified from the CELM protein fraction using the corresponding Mon-Pol affinity chromatography resin. Resin-bound CELM proteins were eluted with the corresponding monosaccharide. 2D-SDS-PAGE (pH 4-7) revealed 9 Mannose- and approximately 100 Galactose-selective CELM lectins. In summary, the CELM glycocalyx contains Mannose- and Galactose-binding lectins that may be involved in translating coronary flow into a cardiac parenchymal response.

Enzymatic hydrolysis of luminal coronary glycosidic structures uncovers their role in sensing coronary flow.

Endothelial luminal glycocalyx (ELG) is a multifunctional complex structure made off of a diversity of glycosilated proteins, and glycosaminoglycans (GAG). Coronary ELG may participate as a sensor of coronary flow (CF) to induce inotropic and dromotropic effects. In isolated perfused guinea pig heart we tested the role of glycosidic groups of glycans bound to proteins and GAG of the ELG on CF-induced inotropic and dromotropic effects. To study the role of saccharide related groups of certain glycans, they were removed by selective enzyme hydrolysis or bound to a selective plant lectin. CF-induced positive inotropic and positive dromotropic control curves were obtained and the effects of intracoronary infusion of enzyme or lectin determined. The analyzed groups were as follow: 1) Fucosidase enzyme and Ulex europeasus lectin; hydrolysis and binding respectively (H&Br) to alpha-linked fucosyl related groups. 2). Endoglycanase-H and Lycopersicon esculentum (H&Br to N-linked beta-1,3GlcNAc related groups). 3) O-glycanase and Arachis hypogea (H&Br to O-linked beta-Gal1, 3GalNac related groups). 4) Sialidase and Maackia amurensis (H&Br to neuraminic acid related groups). In treatments 1-3 both. lectin and corresponding enzyme, equally depressed CF-positive dromotropic effects without affecting positive inotropic effects. In treatment 4 both lectin and enzyme equally depressed CF-positive inotropic effects without dromotropic effects. The differential role of GAG hyaluran or heparan groups on CF-positive inotropism and positive dromotropism respectively was shown. Infusing hyaluranidase removed hyaluran that solely inhibited CF- inotropism while removal of heparan with heparinase solely inhibited CF-dromotropism. Only the effects of hyaluronidase were reversed infusing hyaluronidate. Our results indicate glycans of ELG are elements of complex multimolecular sensors of coronary flow.