Endothelial pannexin 1–TRPV4 channel signaling lowers pulmonary arterial pressure in mice
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Curated by eLife
Evaluation Summary:
This study, which makes a connection between several proteins known to regulate endothelial function in pulmonary arteries, may be of interest to vascular, pulmonary and ion channel physiologists. The study provides compelling evidence that ATP released from pulmonary artery endothelial cell (EC) pannexin1 channels activates TRPV4 channels via an EC P2Y2R-PLC-DAG-PKC alpha pathway that is facilitated by the scaffolding protein Caveolin-1 and that this pathway helps to maintain low pulmonary vascular resistance and pulmonary artery pressure. Identification of this pathway provides new drug targets to improve pulmonary endothelial function in disease states such characterized by impaired endothelial function. What remains to be established or understood is the physiological stimulus for activation of the pannexin1 channels and ATP release and also the potential dark-side of overactivity of EC TRPV4 channels, which appear to have negative effects on EC function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1–ATP–TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1–P2Y2R–TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.
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Author Response:
Joint Public Review:
Using an impressive combination of endothelial-specific knockouts, the investigators provide strong evidence for the following signaling pathway in pulmonary arteries: Activation of Pannexin1 to the release of ATP-activation to P2Y receptors activation of PKC to activation of TRPV4 channels, anchored by caveolin1.
The study by Daneva et al examines the link between Cav-1, Panx1, P2Y2R and PKC in modulating TRPV4 channel activity. The authors hypothesize that activation of this signaling pathway, and specifically TRPV4, controls the reactivity of pulmonary arteries and contributes to fine-tuning pulmonary arterial pressure. To examine this hypothesis, the authors deployed an impressive number of techniques that include several endothelial-specific knockouts of key members of the signaling …
Author Response:
Joint Public Review:
Using an impressive combination of endothelial-specific knockouts, the investigators provide strong evidence for the following signaling pathway in pulmonary arteries: Activation of Pannexin1 to the release of ATP-activation to P2Y receptors activation of PKC to activation of TRPV4 channels, anchored by caveolin1.
The study by Daneva et al examines the link between Cav-1, Panx1, P2Y2R and PKC in modulating TRPV4 channel activity. The authors hypothesize that activation of this signaling pathway, and specifically TRPV4, controls the reactivity of pulmonary arteries and contributes to fine-tuning pulmonary arterial pressure. To examine this hypothesis, the authors deployed an impressive number of techniques that include several endothelial-specific knockouts of key members of the signaling pathway, optical and electrical patch clamping, MRI and in situ proximity ligation assay (PLA). The data seem of high quality and for the most part, supportive of the conclusions of the study. The results may have broad implications for regulating pulmonary artery regulation and potential identification of novel targets to treat pulmonary artery dysfunction.
- The physiological role of the proposed pathway is unclear. PAP is normally low (8 - 20 mm Hg). Are the authors proposing that this pathway is always engaged to maintain low PAP? If so, then how is Pannexin 1 being tonically activated? This would also imply that there exists a tonic constrictor pathway which Pannexin1-TRPV4 opposes. Does this exist? Or the proposed Panx1-V4 pathway only engaged in the face of pulmonary hypertension. It is hard to envision a dilatory pathway when the system is already at low pressure, i.e., relaxed.
We appreciate the feedback from the Reviewers and the Editor. It has generally been considered that PAs, due to low intraluminal pressure, are relaxed/low-resistance. However, this assumption results from the lack of detailed studies on pressure-induced (myogenic) constriction in small PAs under resting conditions. In this manuscript we provide evidence that small PAs (50-100 microns) show myogenic constriction (Fig. 2B, 2D and 3G), but large PAs (> 200 microns) do not (Supplemental Fig. 2A). We also show that PAs from endothelial Panx1-/-, TRPV4-/-, and P2Y2R-/- mice develop significantly higher myogenic constriction compared to PAs from the respective control mice (Fig. 2B, 2D and 3G). These data strongly support tonic activation of endothelial Panx1–P2Y2R–TRPV4 channel pathway and its dilatory effect under basal conditions. In addition to myogenic constriction, agonist-induced constriction was also higher in PAs from endothelial Panx1-/-, TRPV4-/-, and P2Y2R-/- mice compared to the control mice (Fig. 2C, 2E, and 3H). The detailed studies of myogenic constriction of PAs and mechanisms involved will be published in a separate manuscript.
As the reviewer pointed out, it is plausible that the Panx1-dependent signaling is altered in pulmonary hypertension (PH), a possibility that has not been tested. In this regard we have shown that endothelial TRPV4 channel activity is impaired in PAs from PH patients and mice models of PH1.
- The use of the term, "small, resistance-sized pulmonary arteries" is curious. Pulmonary arteries have low resistance and pressure. What is the basis of using this term?
While the general opinion is that PAs are low-resistance or are in a completely relaxed state, there are no detailed studies showing a lack of myogenic constriction in pressurized small PAs under basal conditions. Our new PA pressure myography data show that small PAs (~ 50-100 microns) develop pressure-induced/myogenic constriction, whereas large PAs (~ 200 microns or more) do not (Fig. 2B, 2D, and 3G, Supplemental Fig. 2A). We use the term “resistance-sized PAs” to describe PAs that show myogenic constriction (~ 50-100 microns, used in this study). We present evidence that PAs develop myogenic constriction at the physiological intraluminal pressure (15 mm Hg, Fig. 2B, 2D, and 3G). We also show that PAs from endothelial Panx1-/-, TRPV4-/-, and P2Y2R-/- mice develop significantly higher myogenic constriction compared to PAs from the respective control mice. Thus, endothelial knockout of Panx1, P2Y2R, or TRPV4 channel increases PA contractility and elevates PAP.
- The major concern is related to conceptual significance. The reviewer appreciates that the work presented here connects Cav-1, Panx1, P2Y2R, PKC and TRPV4 into a signaling axis regulating pulmonary artery reactivity. However, this group has already published similar papers implicating a role for this axis in pulmonary arteries (and a similar axis in systemic arteries), and comparable conclusions have been reached by examining members of the pathway independently. Therefore, it is unclear what new conceptual information is gained, other than the link between all the proteins in the complex. Perhaps the authors could highlight more the major gaps in knowledge and novel aspects of their work.
We thank the reviewers and the Editor for identifying the strengths of the manuscript and for their constructive feedback. We recently reported that endothelial TRPV4 channels decrease the contractility of small pulmonary arteries (PAs) and lower resting pulmonary arterial pressure (PAP) . Moreover, exogenous ATP activated endothelial TRPV4 channels to dilate PAs. However, the regulation of TRPV4 channel activity by endogenously released ATP, the source of endogenously released ATP, and the precise signaling mechanisms for ATP activation of endothelial TRPV4 channels were not known. In the current manuscript, we present a novel signaling axis whereby ATP efflux through endothelial Pannexin 1 (Panx1) activates nearby TRPV4 channels via purinergic receptor signaling to lower PA contractility and PAP. Following key findings contribute to the high conceptual significance and novelty of the study:
First evidence, using endothelial knockout mice, that ATP efflux through endothelial Panx1 lowers PA contractility and PAP. Notably, previous studies have shown that endothelial Panx1 activity does not contribute to vasodilation in systemic arteries and systemic blood pressure regulation.
First direct evidence that ATP efflux through Panx1 promotes endothelial TRPV4 channel activity in PAs, but TRPV4 channel activity does not regulate ATP efflux through Panx1 under resting conditions.
First evidence that ATP effluxed through endothelial Panx1 stimulates purinergic P2Y2 receptor (P2Y2R) signaling to activate TRPV4 channels and lower PA contractility and resting PAP.
Earlier, we showed that endothelial caveolin-1 (Cav-1) lowers the resting PAP. In the current manuscript, we provide evidence that endothelial Cav-1 provides a signaling scaffold for Panx1, P2Y2R, and TRPV4 channels, ensuring their spatial proximity in PAs. Activation of the endothelial Panx1–P2Y2 receptor–TRPV4 channel pathway, enabled by the Cav-1 scaffold, lowers PA contractility and PAP.
PAs are a high-flow vascular bed, yet flow-induced endothelial signaling is poorly understood in PAs. We provide evidence that physiological flow/shear stress increases luminal ATP release through endothelial Panx1 activation.
We have now modified the Introduction and other sections of the manuscript to highlight the conceptual significance and novelty of the results presented in this manuscript.
- The major strengths of the study include the use of EC-specific conditional knockouts of Panx1, TRPV4 and P2Y2R that allowed them to focus on the role played by these protein in the endothelium; the state-of-the-art measurement of TRPV4 Ca2+ sparklets and TRPV4 currents; the use of pressure myography to close-the-loop between the ex vivo studies of TRPV4 sparklets and their in vivo measurement of Right ventricular systolic pressure (RVSP as a surrogate for PAP); their measurement of Right heart mass and function to exclude major effects on heart function as a cause of the observed increase in RVSP; and the use of transfected HEK293 cells to examine the role played by caveolin-1 in the signaling pathway.
Thank you for identifying the strengths of our manuscript. We previously reported that endothelial TRPV4 sparklets dilate PAs via eNOS activation. Specifically, TRPV4 channel activation increased NO levels, an effect that was absent in PAs from eNOS-/- mice. Moreover, TRPV4 channel-induced vasodilation was abolished by NOS inhibitor L-NNA. Also, in endothelial TRPV4-/- mice, endothelial NO levels were reduced . We have now cited these studies. We also show that physiological flow/shear stress activates ATP efflux through endothelial Panx1.
- The results shown support the authors hypothesis and provide new drug and molecular targets to modulate pulmonary vascular resistance, particularly in disease states where endothelial function is compromised.
We agree that our data provide multiple targets for lowering pulmonary artery contractility and pulmonary arterial pressure, including Panx1, P2Y2 receptors, TRPV4 channels, and Cav-1.
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Evaluation Summary:
This study, which makes a connection between several proteins known to regulate endothelial function in pulmonary arteries, may be of interest to vascular, pulmonary and ion channel physiologists. The study provides compelling evidence that ATP released from pulmonary artery endothelial cell (EC) pannexin1 channels activates TRPV4 channels via an EC P2Y2R-PLC-DAG-PKC alpha pathway that is facilitated by the scaffolding protein Caveolin-1 and that this pathway helps to maintain low pulmonary vascular resistance and pulmonary artery pressure. Identification of this pathway provides new drug targets to improve pulmonary endothelial function in disease states such characterized by impaired endothelial function. What remains to be established or understood is the physiological stimulus for activation of the pannexin1 …
Evaluation Summary:
This study, which makes a connection between several proteins known to regulate endothelial function in pulmonary arteries, may be of interest to vascular, pulmonary and ion channel physiologists. The study provides compelling evidence that ATP released from pulmonary artery endothelial cell (EC) pannexin1 channels activates TRPV4 channels via an EC P2Y2R-PLC-DAG-PKC alpha pathway that is facilitated by the scaffolding protein Caveolin-1 and that this pathway helps to maintain low pulmonary vascular resistance and pulmonary artery pressure. Identification of this pathway provides new drug targets to improve pulmonary endothelial function in disease states such characterized by impaired endothelial function. What remains to be established or understood is the physiological stimulus for activation of the pannexin1 channels and ATP release and also the potential dark-side of overactivity of EC TRPV4 channels, which appear to have negative effects on EC function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
-
Joint Public Review:
Using an impressive combination of endothelial-specific knockouts, the investigators provide strong evidence for the following signaling pathway in pulmonary arteries: Activation of Pannexin1 to the release of ATP-activation to P2Y receptors activation of PKC to activation of TRPV4 channels, anchored by caveolin1.
The study by Daneva et al examines the link between Cav-1, Panx1, P2Y2R and PKC in modulating TRPV4 channel activity. The authors hypothesize that activation of this signaling pathway, and specifically TRPV4, controls the reactivity of pulmonary arteries and contributes to fine-tuning pulmonary arterial pressure. To examine this hypothesis, the authors deployed an impressive number of techniques that include several endothelial-specific knockouts of key members of the signaling pathway, optical and …
Joint Public Review:
Using an impressive combination of endothelial-specific knockouts, the investigators provide strong evidence for the following signaling pathway in pulmonary arteries: Activation of Pannexin1 to the release of ATP-activation to P2Y receptors activation of PKC to activation of TRPV4 channels, anchored by caveolin1.
The study by Daneva et al examines the link between Cav-1, Panx1, P2Y2R and PKC in modulating TRPV4 channel activity. The authors hypothesize that activation of this signaling pathway, and specifically TRPV4, controls the reactivity of pulmonary arteries and contributes to fine-tuning pulmonary arterial pressure. To examine this hypothesis, the authors deployed an impressive number of techniques that include several endothelial-specific knockouts of key members of the signaling pathway, optical and electrical patch clamping, MRI and in situ proximity ligation assay (PLA). The data seem of high quality and for the most part, supportive of the conclusions of the study. The results may have broad implications for regulating pulmonary artery regulation and potential identification of novel targets to treat pulmonary artery dysfunction.
The physiological role of the proposed pathway is unclear. PAP is normally low (8 - 20 mm Hg). Are the authors proposing that this pathway is always engaged to maintain low PAP? If so, then how is Pannexin 1 being tonically activated? This would also imply that there exists a tonic constrictor pathway which Pannexin1-TRPV4 opposes. Does this exist? Or the proposed Panx1-V4 pathway only engaged in the face of pulmonary hypertension. It is hard to envision a dilatory pathway when the system is already at low pressure, i.e., relaxed.
The use of the term, "small, resistance-sized pulmonary arteries" is curious. Pulmonary arteries have low resistance and pressure. What is the basis of using this term?
The major concern is related to conceptual significance. The reviewer appreciates that the work presented here connects Cav-1, Panx1, P2Y2R, PKC and TRPV4 into a signaling axis regulating pulmonary artery reactivity. However, this group has already published similar papers implicating a role for this axis in pulmonary arteries (and a similar axis in systemic arteries), and comparable conclusions have been reached by examining members of the pathway independently. Therefore, it is unclear what new conceptual information is gained, other than the link between all the proteins in the complex. Perhaps the authors could highlight more the major gaps in knowledge and novel aspects of their work.
The major strengths of the study include the use of EC-specific conditional knockouts of Panx1, TRPV4 and P2Y2R that allowed them to focus on the role played by these protein in the endothelium; the state-of-the-art measurement of TRPV4 Ca2+ sparklets and TRPV4 currents; the use of pressure myography to close-the-loop between the ex vivo studies of TRPV4 sparklets and their in vivo measurement of Right ventricular systolic pressure (RVSP as a surrogate for PAP); their measurement of Right heart mass and function to exclude major effects on heart function as a cause of the observed increase in RVSP; and the use of transfected HEK293 cells to examine the role played by caveolin-1 in the signaling pathway.
The only weaknesses are: the lack of experimental evidence in this study that eNOS and NO are the downstream effectors of the reduced pulmonary vascular reactivity and PAP (although this has been demonstrated in other studies) and the lack of evidence for what is responsible for the activation of EC Panx1 and the release of EC ATP that is key to the process.
The results shown support the authors hypothesis and provide new drug and molecular targets to modulate pulmonary vascular resistance, particularly in disease states where endothelial function is compromised.
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