Leon J. DeLalio

Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation

Inflammatory cell recruitment to local sites of tissue injury and/or infection is controlled by a plethora of signalling processes influencing cell-to-cell interactions between the vascular endothelial cells (ECs) in post-capillary venules and circulating leukocytes. Recently, ATP-sensitive P2Y purinergic receptors have emerged as downstream regulators of EC activation in vascular inflammation. However, the mechanism(s) regulating cellular ATP release in this response remains elusive. Here we report that the ATP-release channel Pannexin1 (Panx1) opens downstream of EC activation by TNF-α. This process involves activation of type-1 TNF receptors, recruitment of Src family kinases (SFK) and SFK-dependent phosphorylation of Panx1. Using an inducible, EC-specific Panx1 knockout mouse line, we report a previously unidentified role for Panx1 channels in promoting leukocyte adhesion and emigration through the venous wall during acute systemic inflammation, placing Panx1 channels at the centre of cytokine crosstalk with purinergic signalling in the endothelium.

AXL-Mediated Productive Infection of Human Endothelial Cells by Zika Virus

RATIONALE The mosquito-borne Zika virus (ZIKV) is now recognized as a blood-borne pathogen, raising an important question about how the virus gets into human bloodstream. The imminent threat of the ZIKV epidemic to the global blood supply also demands novel therapeutics to stop virus transmission though transfusion. OBJECTIVE We intend to characterize ZIKV tropism for human endothelial cells (ECs) and provide potential targets for intervention. METHODS AND RESULTS We conducted immunostaining, plaque assay, and quantitative reverse transcription-polymerase chain reaction of ZIKV RNA to evaluate the possible infection of ECs by ZIKV. Both the African and the South American ZIKV strains readily infect human umbilical vein endothelial cells and human ECs derived from aortic and coronary artery, as well as the saphenous vein. Infected ECs released infectious progeny virus. Compared with the African strains, South American ZIKV isolates replicate faster in ECs and are partially cytopathic, suggesting enhanced virulence of these isolates. Flow cytometric analyses showed that the susceptibility of ECs positively correlated with the cell surface levels of tyrosine-protein kinase receptor UFO (AXL) receptor tyrosine kinase. Gain- and loss-of-function studies further revealed that AXL is required for ZIKV entry at a postbinding step. Finally, small-molecule inhibitors of the AXL kinase significantly reduced ZIKA infection of ECs. CONCLUSIONS We identified EC as a key cell type for ZIKV infection. These data support the view of hematogenous dissemination of ZIKV and implicate AXL as a new target for antiviral therapy.

A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells

The ATP-releasing channel Panx1 is specifically involved in blood pressure regulation by adrenergic signaling. Regulating blood pressure with ATP Blood pressure is dynamically regulated to enable rapid responses to changes in position and physical or emotional stress, such as exercise or anger and fear. Many signals that activate G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) control vascular tone, including norepinephrine (also known as noradrenaline) released by the sympathetic nervous system, which increases blood pressure. Billaud et al. report that the α1 adrenoreceptor (α1AR)—but not the endothelin-1 or serotonin receptor, which are also Gαq-coupled GPCRs and stimulate vasoconstriction—is specifically coupled to activation of the ATP (adenosine 5′-triphosphate)–releasing channel pannexin1 (Panx1). Mice lacking Panx1 in smooth muscle cells were hypotensive, specifically during their active period of the day. Isolated arteries from these mice did not release ATP and contracted less in response to adrenoreceptor stimulation. Thus, ATP release through Panx1 channels specifically contributes to blood pressure regulation by the sympathetic nervous system. Both purinergic signaling through nucleotides such as ATP (adenosine 5′-triphosphate) and noradrenergic signaling through molecules such as norepinephrine regulate vascular tone and blood pressure. Pannexin1 (Panx1), which forms large-pore, ATP-releasing channels, is present in vascular smooth muscle cells in peripheral blood vessels and participates in noradrenergic responses. Using pharmacological approaches and mice conditionally lacking Panx1 in smooth muscle cells, we found that Panx1 contributed to vasoconstriction mediated by the α1 adrenoreceptor (α1AR), whereas vasoconstriction in response to serotonin or endothelin-1 was independent of Panx1. Analysis of the Panx1-deficient mice showed that Panx1 contributed to blood pressure regulation especially during the night cycle when sympathetic nervous activity is highest. Using mimetic peptides and site-directed mutagenesis, we identified a specific amino acid sequence in the Panx1 intracellular loop that is essential for activation by α1AR signaling. Collectively, these data describe a specific link between noradrenergic and purinergic signaling in blood pressure homeostasis.

Emerging concepts regarding pannexin 1 in the vasculature

Pannexin channels are newly discovered ATP release channels expressed throughout the body. Pannexin 1 (Panx1) channels have become of great interest as they appear to participate in a multitude of signalling cascades, including regulation of vascular function. Although numerous Panx1 pharmacological inhibitors have been discovered, these inhibitors are not specific for Panx1 and have additional effects on other proteins. Therefore, molecular tools, such as RNA interference and knockout animals, are needed to demonstrate the role of pannexins in various cellular functions. This review focuses on the known roles of Panx1 related to purinergic signalling in the vasculature focusing on post-translational modifications and channel gating mechanisms that may participate in the regulated release of ATP.

Pannexin channel and connexin hemichannel expression in vascular function and inflammation.

Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.

Pannexin 1 Channels as an Unexpected New Target of the Anti-Hypertensive Drug Spironolactone

Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. Methods and Results: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited &agr;-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. Conclusions: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.

Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (HbαX)

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named Hb&agr;X) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, Hb&agr;X administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II–induced hypertensive states. Hb&agr;X acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O2 binding in red blood cells. In human vasculature, Hb&agr;X blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, Hb&agr;X modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature.

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

TRPV4 (Transient Receptor Potential Vanilloid 4) Channel–Dependent Negative Feedback Mechanism Regulates Gq Protein–Coupled Receptor–Induced Vasoconstriction

Objective— Several physiological stimuli activate smooth muscle cell (SMC) GqPCRs (Gq protein–coupled receptors) to cause vasoconstriction. As a protective mechanism against excessive vasoconstriction, SMC GqPCR stimulation invokes endothelial cell vasodilatory signaling. Whether Ca2+ influx in endothelial cells contributes to the regulation of GqPCR-induced vasoconstriction remains unknown. Ca2+ influx through TRPV4 (transient receptor potential vanilloid 4) channels is a key regulator of endothelium-dependent vasodilation. We hypothesized that SMC GqPCR stimulation engages endothelial TRPV4 channels to limit vasoconstriction. Approach and Results— Using high-speed confocal microscopy to record unitary Ca2+ influx events through TRPV4 channels (TRPV4 sparklets), we report that activation of SMC &agr;1ARs (alpha1-adrenergic receptors) with phenylephrine or thromboxane A2 receptors with U46619 stimulated TRPV4 sparklets in the native endothelium from mesenteric arteries. Activation of endothelial TRPV4 channels did not require an increase in Ca2+ as indicated by the lack of effect of L-type Ca2+ channel activator or chelator of intracellular Ca2+ EGTA-AM. However, gap junction communication between SMCs and endothelial cells was required for phenylephrine activation or U46619 activation of endothelial TRPV4 channels. Lowering inositol 1,4,5-trisphosphate levels with phospholipase C inhibitor or lithium chloride suppressed phenylephrine activation of endothelial TRPV4 sparklets. Moreover, uncaging inositol 1,4,5-trisphosphate profoundly increased TRPV4 sparklet activity. In pressurized arteries, phenylephrine-induced vasoconstriction was followed by a slow, TRPV4-dependent vasodilation, reflecting activation of negative regulatory mechanism. Consistent with these data, phenylephrine induced a significantly higher increase in blood pressure in TRPV4−/− mice. Conclusions— These results demonstrate that SMC GqPCR stimulation triggers inositol 1,4,5-trisphosphate–dependent activation of endothelial TRPV4 channels to limit vasoconstriction.

Small Interfering RNA-Mediated Connexin Gene Knockdown in Vascular Endothelial and Smooth Muscle Cells

Global knockout of vascular connexins can result in premature/neonatal death, severe developmental complications, or compensatory up-regulation of different connexin isoforms. Thus, specific connexin gene knockdown using RNAi-mediated technologies is a technique that allows investigators to efficiently monitor silencing effects of single or multiple connexin gene products. The present chapter describes the transient knockdown of connexins in vitro and ex vivo for cells of the blood vessel wall. In detail, different transfection methods for primary endothelial cells and ex vivo thoracodorsal arteries are described. Essential controls for validating transfection efficiency as well as targeted gene knockdown are explained. These protocols provide researchers with the ability to modify connexin gene expression levels in a multitude of experimental setups.