Author: Peyronnet, Rémi ; Ravens, Ursula ; Grün, Dominic ; Simon‐Chica, Ana ; Schneider‐Warme, Franziska ; Klesen, Alexander ; Kohl, Peter ; Rog‐Zielinska, Eva A. ; Hilgendorf, Ingo ; Chan, Andy ; Lother, Achim ; Greiner, Joachim ; Emig, Ramona

AbstractMacrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical – in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue‐resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch‐induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non‐selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch‐clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left‐ventricular scar tissue 28 days after cryoablation, stretch‐induced current characteristics are not significantly different compared to non‐injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre‐existing cardiac and circulation‐recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage‐traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM.

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Key points

Bone marrow‐derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch‐induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane.

Stretch‐activated current profiles show characteristics of cation non‐selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR.

Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM.

Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR.

Stretch‐induced currents, Piezo1 mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28 days after cryoinjury compared to sham operated mice.

01 Aug 2024·American Journal of Physiology-Cell Physiology

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells

Article

Author: Tseng, Yun Ting ; Yarishkin, Oleg ; Kuhn, Megan ; Križaj, David ; Lakk, Monika ; Rudzitis, Christopher N. ; Stamer, W. Daniel ; Baumann, Jackson M. ; De Ieso, Michael L.

Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

24 May 2024·Circulation Research

NETosis Drives Blood Pressure Elevation and Vascular Dysfunction in Hypertension

Article

Author: Hennen, Elizabeth M. ; Ao, Mingfang ; Patrick, David M. ; Ahmad, Taseer ; Krishnan, Jaya ; de la Visitación, Néstor ; Kirabo, Annet

BACKGROUND:Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction.METHODS: NETosis-deficient Padi4−/− mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence. RESULTS:Padi4−/− mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis. CONCLUSIONS:These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.

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