# Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol

Edyta Glogowska, Gregor Jose, Ana Rita Dias Araújo, Malika Arhatte, Raphael Divita, Coraline Borowczyk, Thibault Barouillet, Baile Wang, Frédéric Brau, Rémi Peyronnet, et al.

<figure><img src="image_1.png" /></figure>

To cite this version:

Edyta Glogowska, Gregor Jose, Ana Rita Dias Araújo, Malika Arhatte, Raphael Divita, et al. Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol. Cell Reports, 2025, 44 (4), pp.115542. 10.1016/j CELREP.2025.115542. hal-05371290

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**Cell Reports**

# Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol

## Graphical abstract

<figure><img src="image_3.png" /></figure>

## Authors

Edyta Glogowska, Gregor P. Jose,
Ana Rita Dias Araújo, ..., Aimin Xu,
Laurent Yvan-Charvet, Eric Honoré

## Correspondence

honore@ipmc.cnrs.fr

## In brief

Glogowska et al. show that pressure-
induced opening of the mechanosensitive
Piezo1 cationic channel in macrophages
is potentiated by a chronic treatment with
oxLDL or 7-ketocholesterol, while acutely
inhibited by DHA. The differential
modulation by pro- and anti-atherogenic
lipids points to the importance of
macrophage Piezo1 in atherosclerosis
development and progression.

## Highlights

* Genetic invalidation of Piezo1 in myeloid cells reduces atherosclerosis development

* Chronic oxLDL or 7-ketocholesterol treatment potentiates Piezo1 currents in macrophages

* 7-Ketocholesterol increases Piezo1 expression and lowers membrane cholesterol/order

* The anti-atherogenic free ω3 polyunsaturated fatty acid DHA acutely inhibits Piezo1

<figure><img src="image_4.png" /></figure>
## Article

# Potentiation of macrophage Piezo1 by atherogenic 7-ketocholesterol

Edyta Glogowska,<fnref n="1" />,<fnref n="5" /> Gregor P. Jose,<fnref n="1" />,<fnref n="5" /> Ana Rita Dias Araújo,<fnref n="1" /> Malika Arhatte,<fnref n="1" /> Raphael Divita,<fnref n="1" /> Coraline Borowczyk,<fnref n="4" /> Thibault Barouillet,<fnref n="4" /> Baile Wang,<fnref n="3" /> Frédéric Brau,<fnref n="1" /> Rémi Peyronnet,<fnref n="2" /> Amanda Patel,<fnref n="1" /> Bruno Mesmin,<fnref n="1" /> Takeshi Harayama,<fnref n="1" /> Bruno Antonny,<fnref n="1" /> Aimin Xu,<fnref n="3" /> Laurent Yvan-Charvet,<fnref n="4" /> and Eric Honoré<fnref n="1" />,<fnref n="3" />,<fnref n="6" />,<fnref n="7" />,*

## SUMMARY

The mechanosensitive ion channel Piezo1 present in endothelial and smooth muscle cells, as well as in macrophages, is emerging as a novel, important player in the etiology of atherosclerosis. Here, we show that myeloid-specific deficiency of Piezo1 in atherogenic Ldlr⁻/⁻ mice reduces plaque formation. Moreover, chronic oxLDL, as well as its main oxysterol 7-ketocholesterol (7-KC), promotes Piezo1 opening by pressure stimulation in both mouse macrophages and transfected HEK cells. 7-KC dramatically enhances Piezo1 current amplitude and slows down inactivation and deactivation. This up-modulation involves an increase in Piezo1 expression, as well as a potentiation of mechanical gating that depends on membrane cholesterol depletion and decreased order. By contrast, Piezo1 is inhibited by the athero-protective free docosahexaenoic acid, either without or with 7-KC. Altogether, these findings indicate that macrophage Piezo1 is differentially modulated by pro- and anti-atherogenic lipids, pointing to the role of Piezo1 and its potentiation by oxysterols in atherosclerosis.

## INTRODUCTION

Atherosclerosis (AS) is a metabolic and inflammatory disease of the large arteries.<fnref n="1" />⁻<fnref n="4" /> Coronary artery disease is the most common type of heart disease caused by AS, which accounts for about 50% of all deaths in Western society.<fnref n="5" /> The main causes for disease development include elevated cholesterol level, high blood pressure, smoking, diabetes, obesity, and a lack of physical activity.<fnref n="2" /> Thus, AS is a major public health issue worldwide, leading to significant morbidity, mortality, and economic burden.<fnref n="5" />

Macrophages centrally contribute to the initiation and progression of plaque formation within the arteries.<fnref n="6" />⁻⁹ Cholesterol by itself is not inherently pro-atherogenic, but its effects depend on its modification and cellular localization. Unmodified cholesterol is primarily esterified and safely stored in intracellular lipid droplets. However, the oxidation of low-density lipoprotein (LDL) cholesterol is a key driver of AS, triggering endothelium dysfunction, foam cell formation, inflammation, and cell death.¹⁰⁻¹² 7-Ketocholesterol (7-KC) is the major oxysterol

formed when LDL particles are oxidized and is recognized as a chronic causative agent in AS.¹⁰⁻¹²

The hemodynamic forces exerted by blood flow, including shear stress, tensile stress, and cyclic strain on the arterial walls have also long been recognized to play a significant role in the initiation of AS.¹³⁻¹⁶ Areas of arteries exposed to disturbed shear stress, such as arterial curvatures and branch points are more prone to AS.¹⁶⁻¹⁹ In contrast, regions with high, unidirectional steady shear stress tend to be protected from plaque formation. The mechanobiology of endothelial cells has been the subject of intense research over the years and its role in AS is well established.¹³⁻¹⁶ However, the role of macrophage mechanotransduction and its possible implication in AS is only starting to emerge.²⁰

Piezo1 is a mechanosensitive (MS) non-selective cationic channel permeable to calcium that is activated in response to a variety of mechanical stimuli including shear stress and pressure, as well as substrate rigidity (for reviews see Murthy et al.²¹⁻²⁴). Notably, Piezo1 is a newly recognized contributor to AS, influencing endothelial inflammation, foam cell formation,

<figure><img src="image_5.png" /></figure>

<footnote n="1">¹Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Institut de Pharmacologie Moléculaire et Cellulaire, Labex ICST, 06560 Valbonne, France</footnote>

<footnote n="2">²Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg - Bad Krozingen, Medical Center - University of Freiburg and Faculty of Medicine, University of Freiburg, Freiburg, Germany</footnote>

<footnote n="3">³State Key Laboratory of Pharmaceutical Biotechnology, Department of Medicine and Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China</footnote>

<footnote n="4">⁴Institut National de la Santé et de la Recherche Médicale, Inserm, Université Côte d'Azur, Centre Méditerranéen de Médecine Moléculaire (C3M), Atip-Avenir, Fédération Hospitalo-Universitaire (FHU) Oncoage, Nice, France</footnote>

<footnote n="5">⁵These authors contributed equally</footnote>

<footnote n="6">⁶Senior author</footnote>

<footnote n="7">⁷Lead contact</footnote>

<footnote>*Correspondence: honore@ipmc.cnrs.fr
https://doi.org/10.1016/j.celrep.2025.115542</footnote>
<figure><img src="image_6.png" /></figure>

(B) Enzymatically digested aortas from these mice were analyzed by flow cytometry to track recruitment of Tomato-positive myeloid cells. Representative dot plots showing the gating strategy used to analyze $dtTomato^+$ macrophages and myeloid cells based on CD64 and FolR2 markers (left) and quantification (right). CD45 was used to identify immune cells. For BM WT myeloid cells one point (23.6) is out of the scale. Data are represented as means ± SEM. A Mann-Whitney U test was used for statistical analysis. \*p < 0.05.

<caption>**Figure 1. Myeloid deficiency of Piezo1 reduces AS development**</caption>

(A) Representative hematoxylin and eosin (H&E)-stained proximal aortas and quantification of atherosclerotic lesion area in aortic sinuses from lethally irradiated *Ldlr<sup>-/-</sup>* mice transplanted with bone marrow (BM) derived from control (LysM<sup>Cre+tdTomatoSTOPfl/+</sup>; BM WT) and myeloid-specific Piezo1-deficient (LysM<sup>Cre+tdTomatoSTOPfl/+</sup> Piezo1<sup>del/lox</sup>; BM KO) mice and fed a Western diet (WD, TD88137, Ssniff) for 12 weeks. Scale bar, 200 µm. Estimated plaque area was decreased by 35% in the KO (left panels). The histogram compares plaque areas between WT and KO transplanted mice (N = 8).

macrophage polarization, matrix metalloproteinase production, and cell death.²⁵⁻³⁶ Since 7-KC, mechanical stress, and Piezo1 activation are all centrally implicated in the etiology of AS, we hypothesized that these important factors may be functionally linked.

Omega-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) have long been known for their protective effect against cardiovascular diseases, including AS.³⁷⁻³⁹ These beneficial effects are attributed at least in part to their anti-inflammatory and lipid-lowering properties. Moreover, DHA alters many basic physical properties of the cell membrane and is thus anticipated to influence cellular mechanosensing.⁴⁰⁻⁴² In this study, we further explored the hypothesis that DHA may exert a protective role against AS through the modulation of Piezo1, possibly by interfering with 7-KC.

Our findings reveal that chronic 7-KC treatment acts as an important endogenous Piezo1 modulator by potentiating both expression and mechanical activation of the channel in native macrophages. Conversely, free DHA potently inhibits pressure-dependent activation of Piezo1, without or with a 7-KC chronic treatment. The differential modulation of macrophage Piezo1 by pro- and anti-atherogenic lipids puts this MS channel upfront in the progression of AS.

## RESULTS

### Myeloid-specific deficiency of Piezo1 in atherogenic Ldlr<sup>-/-</sup> mice reduces plaque development

We bred control (LysM Cre Piezo1<sup>+/+</sup>; wild-type [WT]) and myeloid-specific Piezo1-deficient (LysM Cre Piezo1<sup>del/lox</sup>; KO) mice with a fluorescent reporter tool strain (TomatoSTOP<sup>lox</sup>, A14), transplanted bone marrow from these mice into irradiated *Ldlr<sup>-/-</sup>* mice, and, after a recovery period, placed the recipients on a Western diet (atherogenic) for 12 weeks. Piezo1 deficiency reduced the development of atherosclerotic lesions in proximal aortas (Figure 1A). Moreover, we observed an almost 2-fold decrease in the recruitment of myeloid cells and conversion into macrophages to aortic arches as quantified by flow cytometry using the Tomato reporter (Figure 1B). We excluded a role of Piezo1 on blood cell production as these mice had similar blood

leukocytes (Figure S1A). We observed a similar decrease in the recruitment of macrophages into the peritoneal cavity when Piezo1 was invalidated, although the difference did not reach significance (Figure S1B).

### Piezo1 up-modulation by chronic 7-KC in native mouse macrophages

We studied the effect of a chronic treatment (as occurring in AS) with 10 µM 7-KC for 24 h (LDL-free medium) on cultured mouse bone marrow-derived macrophages (BMDMs) (Figures 2 and S2). A majority of the cells in culture were positive for the macrophage marker F4/80 (Figure S3A, top panel), lipid droplets were visible in the cytosol in the presence of 10 µM 7-KC (Figure S3A, bottom panel), and no cell death was detected, unlike at a concentration of 25 µM (not shown). Using the LysM Cre driver we specifically invalidated the Piezo1 floxed allele in mouse BMDMs (Figure S3B, red bars). MS inward currents were elicited by fast negative pressure pulses of increasing amplitude at a holding potential of -80 mV in the cell-attached patch-clamp configuration (Figures 2 and S2). We observed pressure-activated currents in WT BMDMs with very slow kinetics of activation, none or very mild inactivation ($I_{peak}/I_{steady}$: 1.2), and delayed deactivation, which were activated by the Piezo1 chemical activator Yoda1 (Figures 2A and S2A, left panels, black traces; Figures S3C and S3E-G). Notably, this MS current was greatly enhanced by 7-KC (which was removed from the bath by three intensive washes prior to recordings) in non-polarized M0, as well as in M1 (pro-inflammatory macrophages) or M2 (anti-inflammatory macrophages) polarized BMDMs (Figures 2A-2C and S2A-S2C, red traces, left panels; Figures S3D, S3H, and S3L). Activation kinetic was not altered, while inactivation and deactivation further slowed down in the presence of 7-KC (Figures S3E-S3G, S3I-S3K, and S3M-S3O). In BMDMs, we observed some baseline opening of Piezo1 visible at 0 mm Hg. This was even more evident either in the presence of Yoda1 (Figure S3C) or after chronic treatment with 7-KC (Figure 2, red traces, left panels). The opening of Piezo1 at rest in macrophages is likely due to poor inactivation in BMDMs (presumably related to MyoD-family inhibitor protein expression⁴³ and to a shift of pressure-dependent activation, as previously
<figure><img src="image_7.png" /><figcaption>Figure 2. Piezo1 potentiation by chronic 7-KC in native mouse BMDMs</figcaption></figure>

(A) Mechanosensitive inward mean currents recorded in the cell-attached configuration at holding potential −80 mV in bone marrow differentiated macrophages (BMDMs) from WT and KO mice (non-polarized, M0) and elicited by fast negative pressure pulses of Δ 10 mm Hg (bottom traces). After 7 days of differentiation with M-CSF, cells were treated for 24 h in LDL-free medium with EtOH (vehicle, in black) or 10 µM 7-KC (in red). Right inset: histogram of peak current amplitude (maximal current at −60 mm Hg) in EtOH (in black) or 7-KC (in red) condition, comparing WT and KO macrophages.

(B) Mean currents elicited by membrane stretch (bottom traces) and recorded in the cell-attached configuration at −80 mV in BMDMs polarized to M1. BMDMs M0 cells were first polarized to M1 in complete medium supplemented with LPS (10 ng/mL) and IFN-γ (20 ng/mL) cocktail for 24 h, and then treated with either EtOH (black traces) or 10 µM 7-KC (in red) for an additional 24 h in LDL-free medium. Left inset: M1 polarization was confirmed by TNFα gene expression and is represented as relative expression to TOP1. Right inset: peak current amplitude in EtOH (in black) or 7-KC (in red) condition, comparing WT and KO macrophages.

(C) Mean currents elicited by membrane stretch (bottom traces) and recorded in the cell-attached configuration at −80 mV in BMDMs polarized to M2. BMDM M0 cells were first polarized to M2 in complete medium supplemented with IL-4 (20 ng/mL) and IL-13 (20 ng/mL) cocktail for 24 h, and then treated with either EtOH (black traces) or 10 µM 7-KC (in red) for an additional 24 h in LDL-free medium. Left inset: M2 polarization was confirmed by CD206 gene expression and is represented as relative expression to TOP1. Right insets: peak current amplitude in EtOH (in black) or 7-KC (in red) condition, comparing WT and KO macrophages. *n*, number of individual recordings are shown above the traces. N = 2, number of animals from each genotype used for isolation of bone marrow mesenchymal stem cells and differentiation to macrophages. Same mice as donor mice in Figure 1. Data are represented as means ± SEM. A Mann-Whitney U test was used for statistical analysis. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.0001.

demonstrated for Yoda1<sup>44</sup>). Additionally, difference in membrane lipid composition<sup>41,42</sup> and/or mechanoprotection by cytoskeletal elements<sup>45</sup> may also be involved. Importantly, the pressure-induced inward current was dramatically decreased upon Piezo1 deletion (KO), without or with 7-KC chronic treatment for 24 h (Figures 2A–2C and S2A–S2C, right panels; Figures S3D, S3H, and S3L). Similar findings were obtained using Fura-2 calcium imaging where the Yoda1 (10 µM) activation was significantly potentiated by a chronic treatment with 10 µM 7-KC (Figure S1C). Of note, we observed that Piezo1 mRNA expression was consistently increased in BMDMs (M0, M1, and M2) chronically treated with 7-KC (Figure S3B, dark gray bars). Similarly, Piezo1 protein expression (total and at the membrane) was significantly enhanced by a 7-KC chronic treatment

in BMDMs (Figures S1D–S1F). These findings indicate that the native MS current in mouse BMDMs is essentially due to Piezo1 opening and can be potentiated by a chronic treatment with 7-KC.

Chronic treatment with 7-KC potentiates native Piezo1 currents in the RAW macrophage cell line

As a cell model system, we further tested the effect of a chronic treatment with oxidized LDL (oxLDL)/7-KC for 24 h on the mouse macrophage cell line RAW (Figures S4 and S5). Cells were cultured either in the absence or presence of 50 µg/mL LDL (Figures S4A, S4B, and S5A, black traces). Again, pressure-induced currents in non-polarized RAW cells (M0) were slowly activating, non- or poorly inactivating, and characterized by a
<figure><img src="image_8.png" /></figure>

remarkably slow deactivation rate (inward current was still prom-
inent 1 s after termination of the pressure pulse; Figures S4A,
S5A, and S6B, black traces). Piezo1 mRNA was detected in
RAW cells and siRNA-mediated knockdown fully suppressed
the pressure-induced current (Figures S6A and S6B). Moreover,
the RAW MS current was reversibly enhanced by Yoda1 (Fig-
ure S6C). Thus, Piezo1 underlies the MS current induced by
pressure stimulation in RAW cells, as previously observed for
BMDMs (Figures 2 and S2). Removing LDL from the serum did
not significantly affect current amplitude or kinetics (Figures
S4B and S6D–S6G). However, when cells were grown for 24 h
in the presence of 50 µg/mL oxLDL (instead of LDL), current
amplitude was significantly enhanced (even though oxLDL was
omitted during the electrophysiological recordings following
three intensive washes; Figures S4A, S4B, S5A, red traces,
and S6D). Activation kinetics was not altered, although inactiva-
tion and deactivation were slower in the presence of oxLDL
(Figures S6E–S6G). Current amplitude elicited by pressure
pulses was again significantly increased after a chronic treat-
ment with 7-KC for 24 h (Figures S4C, S4D, S5B, and S6H). As
previously observed for BMDMs, activation kinetic was unal-
tered, while inactivation and deactivation became slower in the
presence of 7-KC (Figures S6I–S6K). In parallel experiments,
RAW cells were polarized in M1 or M2 state for 24 h prior to a
7-KC chronic treatment (Figures S4E–S4H, S5C, S5D, and
S6L–S6S). Similarly, the native MS current was enhanced by
about 2-fold with 7-KC (red traces) in M1 or M2 polarized RAW
cells (Figures S4E–S4H, S5C, S5D, S6L, and S6P) and both inac-
tivation and deactivation slowed down without a change in acti-
vation rate (Figures S6M–S6O and S6Q–S6S). These findings
obtained with a macrophage cell line further demonstrate that
a chronic treatment with oxLDL or with the oxysterol 7-KC en-
hances the endogenous Piezo1 current amplitude and slows
down inactivation and deactivation kinetics.

oxLDL or 7-KC enhances Piezo1 currents in transfected
HEK Piezo1<sup>del</sup> cells

Next, we explored whether Piezo1 currents may similarly be
up-modulated by oxLDL/7-KC in transiently transfected HEK
Piezo1<sup>del</sup> that lack native Piezo1 channels (Figure 3). Using
this exogenous system, we avoid any possible effect on
Piezo1 endogenous expression. In agreement with previous
reports,⁴⁶ the exogenous Piezo1 currents were characterized
by faster kinetics of activation and inactivation and lacked
slow deactivation compared with the native Piezo1 currents
recorded in macrophages (Figure 3A, black traces). Again,
removing LDL did not affect Piezo1 currents (Figures 3B and
S7). However, chronic treatment with oxLDL enhanced Piezo1
current amplitude with a slowing down of the inactivation rate
(oxLDL was absent during the electrophysiological recordings)
(Figures 3A and 3B, red traces; Figure S7). Similarly, 7-KC
produced a comparable effect that was visible in the range
of 2.5–10 µM 7-KC (Figures 3C, 3D, and S7). This stimulatory
effect of chronic 7-KC observed in the cell-attached configu-
ration was also visible in the excised inside-out configuration
(up to 5-fold at 10 µM 7-KC, Figure 3E). We tested the effect
of an acute application of 7-KC on Piezo1 currents recorded in
inside-out patches (Figure 3F). Although Yoda1 consistently

induced a prominent activation of the Piezo1 current in these
patches, acute 7-KC application up to 25 µM failed (n = 7) to
affect Piezo1 opening (Figure 3F). Thus, 7-KC behaves like a
Piezo1 activator (i.e., enhancing pressure-induced currents),
although it requires a chronic treatment, unlike Yoda1, which
works acutely and reversibly. Next, we explored whether
7-KC may affect Piezo1 protein expression and/or trafficking
to the plasma membrane in transfected HEK Piezo1<sup>del</sup> cells
(Figures 3G and 3H). We took advantage of a C-terminal
GFP Piezo1 fusion protein for imaging Piezo1 at the plasma
membrane by confocal microscopy.⁴⁷ The membrane/total
Piezo1 fluorescence was unaltered by 7-KC, indicating that,
at least in transfected HEK cells, the increase in Piezo1 cur-
rent is independent of protein expression or trafficking
(Figures 3G and 3H). Thus, the effect of 7-KC on exogenous
Piezo1 expressed in transfected HEK cells may involve an ef-
fect on either single-channel conductance and/or channel
gating. Importantly, the single-channel current amplitude
was not modified by 7-KC (Figure 3I), suggesting that the me-
chanical gating of Piezo1 is likely to be involved in the up-
modulation by 7-KC in HEK cells.

7-KC chronic treatment causes a decrease in
membrane cholesterol

Previous findings indicate that membrane cholesterol greatly
influences Piezo1 mechanical gating.⁴¹,⁴⁸ Depletion of mem-
brane cholesterol with methyl-β-cyclodextrin (MβCD) induces
a large increase in Piezo1 current amplitude, with a major
slowing down of its inactivation (Figures 5C–5E). Since 7-KC
is known to exert a strong negative feedback on 3-hydroxy-
3-methyl-glutaryl-coenzyme A reductase (HMGCR) (that is,
the rate-limiting enzyme required for the endogenous synthe-
sis of cholesterol) and is also expected to compete with and
displace membrane cholesterol, we explored whether a
change in membrane cholesterol may be implicated in the
up-modulation of Piezo1 by 7-KC (Figure 4). First, using
high-performance thin-layer chromatography (HPTLC) we
verified global cholesterol levels (sterol [ST]) in RAW cells
grown in the absence of LDL, without (EtOH) or with 10 µM
7-KC for 24 h, similar to the protocol used for electrophysi-
ology. Cells were extensively washed 3 times with PBS prior
to lipid extraction to eliminate lipids (including 7-KC) from
the culture medium. We observed a net decrease in total
cholesterol (ST) in cells treated with 7-KC, while conversely
cholesteryl ester (STE) was increased (Figures 4A and S8A).
7-KC and presumably 7-KC ester (Figures 4A and S8A; *)
were also visible, indicating that cells accumulate 7-KC, which
becomes esterified. The density plot (Figure 4A, right panel)
revealed that the peak area of 7-KC matches about the area
loss for cholesterol, suggesting that one sterol substitutes
for the other. Accordingly, we hypothesized that 7-KC may
displace and substitute for cholesterol at the plasma mem-
brane. Next, to visualize membrane cholesterol more directly,
we took advantage of the dehydroergosterol (DHE) probe, an
intrinsically fluorescent sterol with absorption/emission in the
ultraviolet (UV) region and properties similar to cholesterol.⁴⁹
We used UV-sensitive low-light-level wide-field imaging to
examine the effect of 7-KC on plasma membrane DHE in living
<figure><img src="image_9.png" /><figcaption>**Figure 3.** oxLDL and 7-KC enhance Piezo1 currents in transfected HEK Piezo1<sup>del</sup> cells</figcaption></figure>

(A) Mechanosensitive inward mean currents recorded in the cell-attached configuration at holding potential –80 mV in HEK Piezo1 knockout cells (HEK Piezo1<sup>del</sup>) exogenously transfected with Piezo1 together with mCherry and elicited by fast negative pressure pulses of Δ 10 mm Hg (bottom traces). Cells were chronically treated with either 50 µg/mL LDL (left panel, in black) or 50 µg/mL oxLDL (right panel, in red) and cultured for 24 h in LDL-free medium.

(B) Pressure-effect curves of inward currents recorded at –80 mV in HEK Piezo1<sup>del</sup> from LDL-free (black symbols), LDL (empty symbols), and oxLDL (red symbols). P<sub>0.5</sub> values (in mm Hg) are –66.9, –59.2, and –50.3, respectively. k values are 17.7, 15.5, and 17.1, respectively. n values are 10, 27, and 26, respectively.

(C) Mean currents elicited by membrane stretch (bottom traces) and recorded in the cell-attached configuration at –80 mV in HEK Piezo1<sup>del</sup> transfected with Piezo1 and mCherry. Cells were treated for 24 h in LDL-free medium with EtOH (vehicle, black traces) or 7-KC (10 µM in red). Inset: lack of a stretch-activated mean current confirmed in mCherry only transfected HEK Piezo1<sup>del</sup> and treated with 10 µM 7-KC for 24 h.

(D) Pressure-effect curves of inward currents recorded at –80 mV in HEK Piezo1<sup>del</sup> from control EtOH-(in black) and 7-KC-treated cells (2.5 µM in orange, and 10 µM in red). P<sub>0.5</sub> values (in mm Hg) are –44.1, –55.6, and –58.8, respectively. k values are 7.7, 11.6, and 18.7, respectively. n values are 47, 22, and 50, respectively. Data are represented as mean ± SEM. A Mann-Whitney U test was used for statistical analysis. \*p < 0.05, \*\*\*p < 0.0001. n, number of individual recordings are shown above the traces.

(E) Inside-out Piezo1 currents elicited by pressure pulses at a holding potential of –80 mV in the presence of EtOH or after a chronic treatment for 24 h with 7-KC (10 µM).

(F) Lack of effect of acutely applied 7-KC (5, 10, and 25 µM), while Yoda1 (5 µM) enhances pressure-induced Piezo1 currents recorded at –80 mV in response to a repetitive –40 mm Hg pressure pulse (inset).

(G) Membrane expression of Piezo1-GFP after a chronic treatment with either EtOH or 10 µM 7-KC for 24 h. Scale bars, 5 µm.

(H) Membrane/total fluorescence in the presence of either EtOH or 7-KC.

(I) Single Piezo1 channel current amplitude after a chronic treatment with either EtOH or 10 µM 7-KC for 24 h recorded at –80 mV without pressure stimulation (representative traces, left panel, and histogram, right panel). Mann-Whitney U test was used for statistical analysis. Data are represented as means ± SEM. The difference is not significant.

RAW cells that were cultured in LDL-free medium, as previously described. After a loading pulse (complexed to MβCD for 2 min) of DHE, cells were extensively washed and imaged after 8 h of chronic treatment with or without 10 µM 7-KC. In control experiments, we verified the absence of DHE fluorescence quenching in the presence of 7-KC (STAR Methods). Notably, 7-KC significantly decreased the DHE fluorescence signal (by about 50%–60%) at the plasma membrane of unpo-

larized M0, and M1- or M2-polarized RAW cells (Figures 4B–4E). Similarly, 7-KC decreased DHE membrane fluorescence of HEK cells by about 70%–80%, either without or with Piezo1 exogenous expression (Figures S8B–S8D). Similar findings were obtained using BODIPY cholesterol instead of DHE (not shown). Altogether, these findings indicate that 7-KC chronic treatment of RAW or HEK cells causes a net reduction in membrane cholesterol.
<figure><img src="image_10.png" /><figcaption>A</figcaption></figure>

*suspected 7-KC ester

<figure><img src="image_11.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Figure 4. 7-KC chronic treatment decreases membrane cholesterol</figcaption></figure>

(A) Fractionation of total lipids from RAW M0 ex-
tracts. Each lane corresponds to a biological
replicate that was grown in LDL-free medium, in
the presence of EtOH (vehicle) or 10 µM 7-KC
(n = 3). On the right are presented two aligned
chromatograms that correspond to the mean ±
SEM of replicates of each tested condition (7-KC
in red). Equal phospholipid masses were applied
onto each sample lane. STE, sterol esters; ST,
sterols; FFA, free fatty acids.

(B) RAW cells were labeled for 2 min at 37°C with DHE/MβCD complex, washed, and chased for 8 h in culture medium (with LDL-depleted serum) in the presence of 7-KC (10 µM) or ethanol (EtOH). Non-polarized RAW M0, and M1 or M2 polarized cells (the same protocol of polarization as for BMDMs, Figure 2) were first labeled with DHE (in green). Wheat germ agglutinin (WGA) was added after the chase to visualize the plasma membrane (in red). The number of macrophages analyzed was for M0: EtOH (n = 25), 7-KC (n = 37); M1: EtOH (n = 40), 7-KC (n = 31); M2: EtOH (n = 37), 7-KC (n = 39). Data are represented as means ± SEM. A Mann-Whitney U test was used for statistical analysis with ****p < 0.0001.

(C-E) Fluorescence microscopy images of RAW cells illustrating the effect of 7-KC on DHE localization. Scale bars, 10 µm.

Potentiation of Piezo1 by 7-KC is prevented by high
cholesterol, while mimicked by membrane cholesterol
depletion

Since the above observations indicate that membrane choles-
terol is substantially lowered by 7-KC, we investigated whether
or not cholesterol enrichment may revert the activation of Piezo1
by 7-KC (Figures 5A and 5B). Notably in RAW cells, the Piezo1
current was inhibited by cholesterol loading and the potentiation
by 7-KC was prevented (Figure 5A, orange traces). Similar find-
ings were obtained in Piezo1-transfected HEK cells (Figure 5B,
orange traces). Conversely, the stimulation of Piezo1 current
by 7-KC was mimicked by membrane cholesterol depletion us-
ing MβCD (Figures 5C-5E). However, the magnitude of potenti-
ation by MβCD was much stronger, especially when considering
the slowing down of the inactivation rate (Figure 5E). Thus, be-
sides a decrease in membrane cholesterol (Figure 4) our electro-
physiological observations (Figures 5C-5E) are consistent with
the possibility that Piezo1 activation by 7-KC may involve an
additional mechanism that would be at play in parallel with mem-
brane cholesterol depletion.

Chronic treatment with 7-KC lowers membrane order, which correlates with Piezo1 potentiation

Cholesterol is an abundant lipid (typical concentrations of about 20–30 mol %) that is known to increase membrane order (i.e., lipid packing)⁵⁰ (graphical abstract). We took advantage of the NR12S probe with confocal microscopy to evaluate the eventual impact of 7-KC on membrane order in BMDMs, RAW, and HEK

cells (Figure 6). This solvatochromic photostable plasma mem-
brane-targeting dye exhibits, in a liquid ordered (L<sub>O</sub>) phase, a
45–50 nm wavelength shift relative to the liquid disordered (L<sub>D</sub>)
phase. Changes in membrane order are estimated by calculating
the generalized polarization (GP) ratio, a normalized ratio of fluo-
rescence intensity at 550–600 and 600–650 nm ranges.
Figures 6A, 6C, and 6E show typical GP images for BMDMs,
RAW, and HEK cells. Images are shown in pseudo color with
red corresponding to the highest values (ordered) and blue to
the lowest values (disordered). We consistently observed that
7-KC dramatically lowers membrane order in the three cell types,
although with a more dramatic effect seen in HEK cells
(Figures 6A–6F). Thus, insertion of 7-KC in the membrane and
replacement of cholesterol causes a defect in lipid packing
that is anticipated to influence Piezo1 mechanical gating.
Remarkably, we observed a correlative trend between the stim-
ulatory effect of 7-KC on Piezo1 current amplitude and the differ-
ence in GP value caused by 7-KC when comparing BMDMs,
RAW, and HEK cells (Figure S9A). Altogether, these observations
suggest that the drop in membrane cholesterol, its replacement
by 7-KC, and the consequent decrease in membrane order
contribute to potentiate Piezo1 mechanical gating.

Free DHA inhibits Piezo1 without or with 7-KC

The cardiovascular beneficial effects of omega-3 PUFAs,
including DHA, have long been recognized.<sup>37-39</sup> Among the
possible mechanisms implicated are membrane effects that
affect ion channel function.<sup>40-42</sup> Here, we hypothesized that
<figure><img src="image_15.png" /><figcaption>Figure 5. Potentiation of Piezo1 by 7-KC is prevented by high cholesterol</figcaption></figure>

(A) Effect of membrane cholesterol enrichment (orange traces) on mean inward currents in RAW M0 cells recorded in the cell-attached patch configuration at –80 mV and elicited by increasing negative pressure pulses (bottom black traces). Cells were chronically treated for 24 h in LDL-free medium either with EtOH (vehicle) or 10 µM 7-KC and then incubated in the external solution (without serum) containing 120 µg/mL water-soluble cholesterol for 45 min at 37°C. Cells were extensively washed 3 times before recording.

(B) Same for HEK Piezo1<sup>del</sup> transfected with Piezo1.

(C) Effect of membrane cholesterol depletion (MβCD, 5 mM, blue trace) on mean inward currents in Piezo1 transfected HEK Piezo1<sup>del</sup> cells recorded in the cell-attached patch configuration at –80 mV and elicited by negative pressure pulse of –60 mm Hg (bottom black trace).

(D) Peak current amplitude (I peak) and (E) time constants of inactivation (τ) at –60 mm Hg pressure pulse in cell-attached configuration from HEK Piezo1<sup>del</sup> transfected with Piezo1. Two protocols for cell treatment were used: (1) 24 h in LDL-free medium with EtOH (black bars) or 7-KC (10 µM, red bars), (2) 45 min at 37°C in external solution without serum (black bars, control) enriched either with water-soluble cholesterol (120 µg/mL, orange bars) or MβCD (5 mM, blue bars). Data are represented as means ± SEM. A Mann-Whitney U test was used for statistical analysis. \*p < 0.05, \*\*\*p < 0.001, \*\*\*\*p < 0.0001. n, number of individual recordings are shown above the traces.

the atheroprotective DHA may exert some of its beneficial effects through the modulation of Piezo1 and could potentially interfere with 7-KC-induced Piezo1 potentiation. We consistently observed that acute DHA application caused a reversible inhibition of native Piezo1 currents recorded in both BMDMs and RAW (Figures 7A and 7B). Similar findings were obtained when Piezo1 was expressed in transfected HEK cells (Figure 7C). The inhibitory effect of acute DHA application was observed both in the excised inside-out and outside-out configurations (Figures 7C and 7D) and occurred at both negative and positive membrane potentials (Figure 7E). The saturated fatty acids did not modify Piezo1 and the mono-unsaturated fatty acid oleic acid only showed a partial inhibition (Figure 7F). Moreover, esterified DHA (DHA methyl ester or DHA triglyceride) was inactive (Figure 7F). Half-maximal reversible inhibition was observed at about 2 µM DHA (Figure 7G). In parallel, we tested the effect of 10 µM DHA on membrane order in HEK cells (that show the most robust decrease in GP value upon 7-KC treatment; Figures 6E and 6F), either in the absence or presence of 7-KC (Figure S9B). We observed no change in membrane lipid order caused by DHA, without or with 7-KC (Figure S9B). Thus, the inhibitory DHA effect appears to be unrelated to an effect on membrane order, unlike the potentiation of Piezo1 by 7-KC (Figures S9A and S9B). In HEK cells grown in the absence of LDL we further tested the ef-

fect of acutely applied increasing concentrations of DHA on Piezo1 currents with or without potentiation by 7-KC (Figure 7C). We consistently observed a reversible inhibition of Piezo1 currents at both 5 and 10 µM DHA, independently of the prior 7-KC chronic treatment (Figure 7C, right graph). Finally, we compared the effect of acute or chronic treatment with DHA on Piezo1 currents in HEK cells (Figures S9C–S9F). DHA (100 µM) was complexed with lipid-free BSA (ratio 5:1), to minimize the amount of free fatty acid. Currents were recorded following three intensive washes of the cells to remove DHA/BSA. With a chronic DHA treatment, we did not observe current inhibition, although the kinetics of inactivation became slower, confirming earlier reports by others<sup>41,42</sup> (Figures S9C–S9F).

DISCUSSION

7-KC is the main oxysterol contained in oxLD implicated in AS.<sup>10–12</sup> 7-KC is taken up by macrophages through scavenger receptors (including CD36) leading to foam cell formation.<sup>51</sup> 7-KC causes a variety of additional effects, including impairment of macrophage migration, inhibition of phagocytosis, inflammation, and induction of cell death.<sup>11,52–55</sup> Thus, 7-KC critically contributes to the development and progression of atherosclerotic plaques.<sup>12</sup>
<figure><img src="image_16.png" /><figcaption>Figure 6. 7-KC causes a decrease in membrane order</figcaption></figure>

Cells were incubated with 10 µM 7-KC/EtOH for 24 h (LDL-depleted serum) followed by NR12S staining and imaged using confocal microscopy. (A, C, and E) The GP maps of BMDMs, RAW, and HEK Piezo1<sup>del</sup> cells treated with EtOH (top panel) and 7-KC (bottom panel), respectively. Scale bars, 5 µm. (B, D, and F) The graphical representation of GP values (EtOH, black bars; 7-KC, red bars). The number of BMDMs analyzed were: M0 EtOH (n = 20), M0 7-KC (n = 31), M1 EtOH (n = 19), M1 7-KC (n = 31), M2 EtOH (n = 41), M2 7-KC (n = 43). The number of RAW macrophages analyzed were: M0 EtOH (n = 30). M0 7-KC (n = 32), M1 EtOH (n = 22), M1 7-KC (n = 20), M2 EtOH (n = 27), M2 7-KC (n = 28). The number of Piezo1 transfected HEK Piezo1<sup>del</sup> cells were: EtOH (n = 39) and 7-KC (n = 39). Data are represented as means ± SEM. A Mann-Whitney U test was used for statistical analysis with ****p < 0.0001.

This study indicates that Piezo1 mechanical activation is potentiated by a chronic treatment with the atherogenic oxLDL or 7-KC, while it is reversibly inhibited by free DHA. The potentiation of Piezo1 to pressure stimulation by 7-KC is paralleled with a reduction in membrane cholesterol and correlated with a decrease in membrane order. Moreover, an increase in Piezo1 mRNA/protein expression mediated by 7-KC is also implicated in BMDMs.

Up-modulation of Piezo1 by depletion of membrane cholesterol in the presence of 7-KC

Using HPTLC analysis, as well as DHE imaging, we observed that 7-KC chronic treatment dramatically decreases membrane cholesterol levels. Since Piezo1 is up-modulated by cholesterol depletion (this study and Glogowska et al.⁴⁸), we hypothesized that such a mechanism may possibly be at play in the potentiation of Piezo1 by 7-KC. How may 7-KC reduce membrane cholesterol levels? First, 7-KC is known to exert a strong negative feedback on HMGCR, thereby decreasing the endogenous synthesis of cholesterol.⁵⁶ Second, at least in macrophages, 7-KC induces the esterification of cholesterol (and of itself: Figures 4A and S8A) leading to the formation of intracellular lipid droplets (Figure S3A, lower panel).⁵¹ Upon activation of acyl CoA:cholesterol acyl transferase in macrophages, a significant proportion of plasma membrane cholesterol was previously

shown to be translocated in the endoplasmic reticulum to be esterified, forming cholesterol esteryls (STE) (Figures 4A and S8A).⁵⁷ Third, an additional mechanism involves a direct competition between 7-KC and cholesterol at the plasma membrane (i.e., 7-KC competing with and displacing membrane cholesterol). Conversely, enrichment of membrane cholesterol effectively prevented the up-modulation of Piezo1 by 7-KC. Thus, these observations are supportive for a possible role of membrane cholesterol depletion in the potentiation of Piezo1 by 7-KC. However, when comparing the effects of 7-KC with MβCD (although it is expected to cause a stronger depletion of membrane cholesterol), the effects were not fully comparable, suggesting that an additional mechanism is likely to be at play.

7-KC causes a decrease in membrane order correlated with Piezo1 potentiation

Assuming that 7-KC substitutes for cholesterol at the membrane, it implies that this oxysterol may be unable to repress Piezo1 opening, as observed for cholesterol (Figures 5A and 5B). The keto function at position 7 in cholesterol presumably causes a tilt of the molecule that may induce surrounding membrane lipid tails to become disordered (graphical abstract), as suggested by the present NR12S data and other experimental evidence.⁵⁸,⁵⁹ Phospholipid tail spacing caused by the insertion of tilted 7-KC is predicted to mimic the effect of membrane
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>Figure 7. Free DHA inhibits Piezo1 in the absence or in the presence of 7-KC</figcaption></figure>

(A) Typical recording of BMDMs M0 in the excised inside-out patch configuration at –80 mV and elicited by a fast negative pressure pulse of –40 mm Hg (bottom black trace). Control (black), acute application of DHA (green), washout (gray).

(B) Same from RAW M0. Inset: histograms of peak current amplitude (DHA at 10 µM, for RAW and BMDMs, respectively).

(C) Same for HEK Piezo1<sup>del</sup> expressing Piezo1 either in the absence of 10 µM 7-KC (left panel) or in the presence of 7-KC (middle panel, red trace). Histogram on the right panel shows peak current amplitude in the different experimental conditions, including 7-KC and DHA, as indicated. In the 7-KC condition (in red, without DHA) three values (1,523; 1,102, and 843 pA) are off scale.

(D) Kinetic of current inhibition by acutely applied DHA (1, 3, 5, and 10 µM) in a HEK Piezo1<sup>del</sup> transfected cell with Piezo1 and recorded in the excised outside-out patch configuration at a holding potential of +60 mV and in response to a +30 mm Hg pressure stimulation.

*(legend continued on next page)*
tension, which drives Piezo1 activation. Thus, it is anticipated
that 7-KC may generate a mechanical pre-stress on the MS
channel, thereby promoting its activation in response to pressure
stimulation and possibly contributing to slowing down its inacti-
vation and deactivation (graphical abstract). We observed that
the decrease in membrane order induced by 7-KC was gradu-
ated when comparing BMDMs, RAW, and HEK cells (that
show the strongest decrease in GP value). Such a difference
may possibly be due to either a lower amount of free cholesterol
in HEK cell membrane compared with macrophages,⁶⁰ or due to
a different organization of membrane lipid domains. Indepen-
dently of the explanation, we observed a correlative trend be-
tween the potentiation of Piezo1 by 7-KC and the effect on the
GP value (i.e., membrane order). This correlation suggests that
the decrease in membrane order induced by 7-KC is contrib-
uting, at least in part, to the up-modulation of Piezo1.

7-KC increases Piezo1 mRNA/protein expression in native mouse macrophages

In BMDMs we also observed that Piezo1 mRNA/protein
expression was consistently increased by 7-KC chronic treat-
ment. This effect was visible either in non-polarized M0 or in
differentiated M1 or M2 macrophages. How 7-KC may influ-
ence Piezo1 mRNA expression remains unclear at this stage.
It is hypothesized that 7-KC may enhance directly or indirectly
the transcription of Piezo1 and/or prevent Piezo1 mRNA
degradation. Thus, although substitution of membrane choles-
terol by 7-KC/decrease in membrane order is thought to cen-
trally influence Piezo1 mechanical gating (as seen in trans-
fected HEK cells), an additional mechanism involving an
enhanced Piezo1 mRNA/protein expression may be another
contributor to the potentiation of Piezo1 by 7-KC in native
macrophages.

Differential regulation of Piezo1 by acute and chronic
DHA treatments

We consistently observed a potent and reversible inhibition of
Piezo1 current by acutely applied DHA in the micromolar range,
both in the inside-out or outside-out patch configurations me-
chanically stimulated with a fast pressure clamp. However,
chronic treatment with DHA did not affect Piezo1 current ampli-
tude, although it slowed down inactivation, as previously
observed by others.⁴¹,⁴² The effect of chronic treatment with
DHA likely involves incorporation into phospholipids by esterifi-
cation, thereby affecting the physical properties of the plasma
membrane.⁴² Importantly, in our study only the free form of
DHA was active, while esterified forms failed to affect Piezo1.
Similar findings were previously observed with the MS K₂P

channels, TREK-1, TREK-2, and TRAAK, although in the oppo-
site direction as in this case DHA typically behaved as a potent
opener of the MS K⁺ channels.⁶¹ We observed that, in the same
patches co-expressing Piezo1 and TREK-1, DHA reversibly in-
hibits Piezo1, while it activates TREK-1 (not shown). Thus, the
reversible and potent inhibition of Piezo1 by free DHA in the
micromolar range appears to be specific for Piezo1. Since acute
treatment with 10 µM DHA does not affect membrane order
(Figure S9B), an alternative mechanism may be involved in
Piezo1 inhibition, including a possible direct interaction with
Piezo1.

Up-modulation of macrophage Piezo1 by 7-KC:
Anticipated role in AS development and progression

Piezo1, which is present in both vascular cells and macro-
phages, is increasingly recognized as an important player in
AS (for reviews see Shinge et al.<sup>28,62</sup>). However, none of these re-
ports identified 7-KC as an endogenous modulator of Piezo1, nor
did they reveal how this MS channel is involved in the disease.
The potential interaction between oxysterols and macrophage
mechanotransduction in the etiology of AS had never been
investigated or even hypothesized before. So far, only small-
molecule synthetic activators of Piezo1 (including Yoda1 and
Jedi1/2<sup>44,63</sup>) were identified, but to our knowledge no circulating
biological modulator promoting Piezo1 opening was ever re-
ported. In this study, we explored the intrinsic mechanism impli-
cated in the up-modulation of Piezo1 by 7-KC in macrophages,
centrally involving a substitution of membrane cholesterol by
7-KC, together with a loss of membrane order. It is anticipated
that 7-KC (in red) is tilted in the membrane due to the presence
of an oxygen (in green) at position 7, in comparison with choles-
terol (in orange; see graphical abstract). The resulting decrease
in membrane order generates a pre-stress on the membrane
(i.e., mimicking tension) that contributes to the potentiation of
Piezo1. This mechanism enhancing Piezo1 mechanical gating
is paralleled with an increase in Piezo1 expression. Importantly,
the atheroprotective agent DHA in its free form acutely inhibits
Piezo1, without or with 7-KC chronic treatment. The differential
modulation of macrophage Piezo1 by pro- and anti-atherogenic
lipids points to the importance of this MS channel in the develop-
ment and progression of AS. Better understanding the role and
regulation of Piezo1 in AS provides valuable insights into new po-
tential therapeutic strategies for the treatment of this devastating
vascular disease.

Limitations of the study

The main mechanistic postulate of this study involving 7-KC up-
modulation of Piezo1 acting through cholesterol displacement

<footnote>(E) Depolarization at positive potentials removes Piezo1 inactivation and allows to record steady-state outward currents upon mechanical stimulation. Typical recording showing reversible DHA inhibition (10 µM, in green) in HEK Piezo1<sup>del</sup> expressing Piezo1 and recorded in the outside-out patch configuration at both +/− 60 mV holding potentials in response to a pressure stimulation of +80 mm Hg.</footnote>

<footnote>(F) Histogram showing Piezo1 inward current inhibition from HEK Piezo1<sup>del</sup> cells transfected with Piezo1 recorded in the inside-out patch configuration at −80 mV in response to a −40 mm Hg pressure stimulation, upon acute application of different fatty acids, including palmitic acid (C16:0), behenic acid (C22:0), oleic acid (C18:1), DHA (C22:6), DHA methyl ester (DHA-ME), and DHA triglyceride (TG) all tested at a concentration of 10 µM. The vehicle ethanol was included in the control solution. Statistical significance (Mann-Whitney U test) is compared with the DHA condition. ***p < 0.0001.</footnote>

<footnote>(G) DHA dose-dependent inhibition of Piezo1 current in inside- and outside-out configuration from HEK Piezo1<sup>del</sup>/Piezo1. Data are represented as means ± SEM.
A Mann-Whitney U test was used for statistical analysis. \*p < 0.05, \*\*p < 0.001, \*\*\*p < 0.0001.</footnote>
and decreased membrane order, together with an increase in Piezo1 mRNA/protein expression in macrophages, remains difficult to test experimentally in vivo. While invalidation of Piezo1 in myeloid cells was shown to significantly reduce plaque formation in a murine AS model (Figure 1), whether or not the specific up-modulation of Piezo1 by oxLDL/7-KC is directly implicated in the development and progression of the disease remains to be demonstrated.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Eric Honoré (honore@ipmc.cnrs.fr).

### Materials availability

This study did not generate new unique reagents. Lysm Cre Piezo1del/lox and Lysm Cre Piezo1lox/lox mouse lines described in this study are available upon request.

### Data and code availability

Raw data are available upon request. Data code availability is not applicable for this study.

## ACKNOWLEDGMENTS

This work was supported by the Agence Nationale de la Recherche (ANR-CE14), the Fondation de France, and the Labex ICST.

## AUTHOR CONTRIBUTIONS

E.H., E.G., B.M., T.H., B.A., A.X., and L.Y.-C. designed the experiments. E.G., G.P.J., A.R.D.A., M.A., R.D., C.B., T.B., B.W., F.B., R.P., A.P., and E.H. performed the experiments. E.H. wrote the manuscript with input from A.P. and L.Y.-C.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND SUBJECT DETAILS

* METHOD DETAILS

* AS Ldlr⁻/⁻ model

* Cell cultures, transient transfection, macrophages polarization

* Identification of macrophages

* Pharmacological treatments

* Preparation of lipoprotein-depleted fetal bovine serum (LDL-free serum)

* siRNAs transfection

* Electrophysiology

* Calcium imaging

* Real time qPCR

* Confocal imaging of Piezo1 membrane expression

* Piezo1 immunofluorescence

* Confocal imaging and calculation of generalized polarization

* DHE/MβCD complex preparation

* DHE labeling protocol

* High performance thin-layer chromatography (HPTLC)

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.115542.

Received: October 24, 2024

Revised: March 13, 2025

Accepted: March 19, 2025

Published: April 11, 2025

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<figure><img src="image_19.png" /></figure>

## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>F4/80 Monoclonal Antibody (BM8) Alexa Fluor™ 488</td><td>ThermoFisher</td><td>RRID: AB_10376287</td></tr><tr><td>CD45-APCCY7</td><td>Biolegend</td><td>RRID: AB_3113064</td></tr><tr><td>CD64-BV786</td><td>BD Biosciences</td><td>RRID: AB_2740644</td></tr><tr><td>FOLR2-APC</td><td>Biolegend</td><td>RRID: AB_2721303</td></tr><tr><td>Piezo-1 Polyclonal Antibody</td><td>ProteinTech</td><td>RRID: AB_2231460</td></tr><tr><td>Goat anti-Rabbit Alexa Fluor™ 488 antibody</td><td>ThermoFisher</td><td>RRID: AB_2576217</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>JM109 competent cells</td><td>Promega</td><td>Cat#L200B</td></tr><tr><td colspan="3"><strong>Biological samples</strong></td></tr><tr><td>Bone marrow differentiated macrophages</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>DMEM, high glucose</td><td>Gibco</td><td>Cat#41965-039</td></tr><tr><td>Gibco™ DPBS, without calcium and magnesium</td><td>Fisher Scientific</td><td>Cat#12559069</td></tr><tr><td>Gibco™ OptiMEM I</td><td>Fisher Scientific</td><td>Cat#15392402</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>HyClone</td><td>SV30143.03</td></tr><tr><td>New born calf serum</td><td>Sigma-Aldrich</td><td>Cat#N4637</td></tr><tr><td>Lipoprotein Deficient Serum</td><td>This paper</td><td>N/A</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>Cat#15140-122</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>Cat#35050-038</td></tr><tr><td>Poly-L-Lysine</td><td>Sigma-Aldrich</td><td>Cat#P4832</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>Cat#25300-054</td></tr><tr><td>Normocin™</td><td>Invivogen</td><td>Cat#ant-nr-1</td></tr><tr><td>Zeocin®</td><td>Invivogen</td><td>Cat#ant-zn-1</td></tr><tr><td>Collagen I</td><td>Gibco</td><td>Cat#A108301</td></tr><tr><td>jetPEI (Polyplus)</td><td>Ozyme</td><td>Cat#101-40N</td></tr><tr><td>Lipofectamine™ RNAiMAX</td><td>Invitrogen</td><td>Cat#13778-150</td></tr><tr><td>Recombinant Mouse M-CSF Protein</td><td>Gibco</td><td>Cat#PMC2044</td></tr><tr><td>Lipopolysaccharides from Escherichia coli O111:B4</td><td>Sigma-Aldrich</td><td>Cat#L2630</td></tr><tr><td>IFN-gamma Protein, Mouse</td><td>MedChemExpress</td><td>Cat#HY-P7071</td></tr><tr><td>IL-4 Protein, Mouse</td><td>MedChemExpress</td><td>Cat#HY-P70644</td></tr><tr><td>IL-13 Protein, Mouse</td><td>MedChemExpress</td><td>Cat#HY-P70460</td></tr><tr><td>Low-Density Lipoprotein (LDL)</td><td>Fisher Scientific</td><td>Cat#10442132</td></tr><tr><td>Oxidized LDL (oxLDL)</td><td>Fisher Scientific</td><td>Cat#15643308</td></tr><tr><td>7-ketocholesterol (7-KC)</td><td>Sigma-Aldrich</td><td>Cat#700015P</td></tr><tr><td>Methyl-β-cyclodextrin (MβCD)</td><td>Sigma-Aldrich</td><td>Cat#C4555</td></tr><tr><td>Cholesterol-Water Soluble</td><td>Sigma-Aldrich</td><td>Cat#C4951</td></tr><tr><td>Yoda1</td><td>Tocris</td><td>Cat#5586</td></tr><tr><td>Invitrogen™ Trypan blue</td><td>Fisher Scientific</td><td>Cat#11584486</td></tr><tr><td>Invitrogen™ Random Primers</td><td>Fisher Scientific</td><td>Cat#10646313</td></tr><tr><td>GoTag(R) G2 DNA Polymerase, 500u</td><td>Promega</td><td>Cat#M7845</td></tr><tr><td>MemGlow™ NR12 S Membrane Polarity Probe</td><td>TebuBio</td><td>Cat#MG08</td></tr><tr><td>Ergosta-5,7,9(11),22-tetraen-3β-ol (DHE)</td><td>Sigma-Aldrich</td><td>Cat#E2634</td></tr><tr><td>β-mercapthoethanol</td><td>Sigma-Aldrich</td><td>Cat#M3148</td></tr></tbody></table>

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Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Red blood cell lysis buffer</td><td>Sigma-Aldrich</td><td>Cat#R7757</td></tr><tr><td>cis-4,7,10,13,16,19-Docosahexaenoic acid (DHA)</td><td>Sigma-Aldrich</td><td>Cat#2534</td></tr><tr><td>Wheat germ agglutinin (WGA; Tetramethylrhodamine conjugate)</td><td>ThermoFisher</td><td>Cat#W849</td></tr><tr><td>BODIPY 493/503</td><td>Fisher Scientific</td><td>Cat#11540326</td></tr><tr><td>Invitrogen™ HCS CellMask™ Stains</td><td>Fisher Scientific</td><td>Cat#10615695</td></tr><tr><td>Potassium bromide</td><td>Sigma-Aldrich</td><td>Cat#746444</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>Cat#D9542</td></tr><tr><td>Hoechst</td><td>Sigma-Aldrich</td><td>Cat#33342</td></tr><tr><td>L-α-lyso-phosphatidylcholine (LPC)</td><td>Avanti Polar Lipids®</td><td>Cat#830071</td></tr><tr><td>sphingomyelin (SM)</td><td>Avanti Polar Lipids®</td><td>Cat#860062</td></tr><tr><td>L-α-phosphatidylcholine (PC)</td><td>Avanti Polar Lipids®</td><td>Cat#840051</td></tr><tr><td>L-α-phosphatidylserine (PS)</td><td>Avanti Polar Lipids®</td><td>Cat#840032</td></tr><tr><td>L-α-phosphatidylinositol (PI)</td><td>Avanti Polar Lipids®</td><td>Cat#840042</td></tr><tr><td>Cardiolipin (CL)</td><td>Avanti Polar Lipids®</td><td>Cat#840012</td></tr><tr><td>L-α-phosphatidylethanolamine (PE)</td><td>Avanti Polar Lipids®</td><td>Cat#840026</td></tr><tr><td>L-α-phosphatidylglycerol (PG)</td><td>Avanti Polar Lipids®</td><td>Cat#841138</td></tr><tr><td>Ceramide (Cer)</td><td>Avanti Polar Lipids®</td><td>Cat#860051</td></tr><tr><td>Mono-Sulfo Galactosyl(β) Ceramide (d18:1/17:0) (MSGCer)</td><td>Avanti Polar Lipids®</td><td>Cat#860572</td></tr><tr><td>Glucosyl(β) Ceramide (d18:1/17:0) (HexCer)</td><td>Avanti Polar Lipids®</td><td>Cat#860569</td></tr><tr><td>C24 Lactosyl(β) Ceramide (d18:1/24:0) (Hex2Cer)</td><td>Avanti Polar Lipids®</td><td>Cat#860577</td></tr><tr><td>5-cholestén-3β-ol (ST)</td><td>Sigma®</td><td>Cat#C8667</td></tr><tr><td>Cholest-5-en-3β-yl heptadecanoate (STE)</td><td>Avanti Polar Lipids®</td><td>Cat#700186</td></tr><tr><td>Cholesteryl formate (ChoF)</td><td>Sigma®</td><td>Cat#S448532</td></tr><tr><td>Arachidonic acid (FFA)</td><td>Sigma®</td><td>Cat#A3611</td></tr><tr><td>1-oleoyl-rac-glycerol (MG)</td><td>Avanti Polar Lipids®</td><td>Cat#330724</td></tr><tr><td>1,3-di(cis-9-octadecenoyl)-glycerol (DG1.3)</td><td>Sigma®</td><td>Cat#D3627</td></tr><tr><td>1,2-dipalmitoyl-sn-glycerol (DG1.2)</td><td>Avanti Polar Lipids®</td><td>Cat#800816</td></tr><tr><td>1,2,3-tri-(9Z-octadecenoyl)-glycerol (TG)</td><td>Sigma®</td><td>Cat#T7140</td></tr><tr><td>HPLC-grade chloroform (CHCl<sub>3</sub>)</td><td>Fisher Scientific</td><td>Cat#10050090</td></tr><tr><td>HPLC-grade methanol (MeOH)</td><td>Sigma®</td><td>Cat#34860</td></tr><tr><td>HPLC-grade hexane</td><td>Honeywell Riedel-de-Haën™</td><td>Cat#34859</td></tr><tr><td>Toluene</td><td>Sigma®</td><td>Cat#244511</td></tr><tr><td>Ethyl acetate (EtAc)</td><td>VWR</td><td>Cat#23880.290</td></tr><tr><td>1-propanol</td><td>Sigma®</td><td>Cat#402893</td></tr><tr><td>Butylhydroxytoluene (BHT)</td><td>Sigma®</td><td>Cat#34750</td></tr><tr><td>Ammonium molybdate tetrahydrate</td><td>Sigma®</td><td>Cat#A7302</td></tr><tr><td>Ascorbic acid</td><td>Sigma®</td><td>Cat#33034</td></tr><tr><td>Copper (II) sulfate pentahydrate</td><td>Sigma®</td><td>Cat#C8027</td></tr><tr><td>Sodium dihydrogen phosphate dihydrate</td><td>VWR Chemicals</td><td>Cat#28011.291</td></tr><tr><td>Perchloric acid 65-71%</td><td>Carlo Erba</td><td>Cat#409193</td></tr><tr><td>Nitric acid 65%</td><td>VWR Chemicals</td><td>Cat#20429.245</td></tr><tr><td>Sulfuric acid 95%</td><td>VWR Chemicals</td><td>Cat#20700.298</td></tr><tr><td>Phosphoric acid</td><td>Sigma®</td><td>Cat#1.00565</td></tr><tr><td>Glass HPTLC Silica gel 60 plates</td><td>Merck/Millipore</td><td>Cat#1.05641.0001</td></tr><tr><td>Collagenase D</td><td>Sigma®</td><td>Cat#01108882001</td></tr><tr><td>Liberase DH Research Grade</td><td>Sigma®</td><td>Cat#5401054001</td></tr><tr><td>Fura-2 a.m.</td><td>ThermoFisher</td><td>Cat#F1221</td></tr><tr><td>Ionomycin</td><td>ThermoFisher</td><td>Cat#I24222</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>Cat#740955.250</td></tr><tr><td>GoScript™ Reverse Transcriptase</td><td>Promega</td><td>Cat#A5000</td></tr><tr><td>qPCR 2X SYBR Green Master Mix</td><td>Eurogentec</td><td>Cat#RT-SN2X-03+NR</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td colspan="3">N/A</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>Human Embryonic Kidney<br>HEK Piezo1<sup>del</sup> cell line</td><td>Laboratory of Ardem Patapoutian<br>(Scripps Research)</td><td>PMID: 28426961</td></tr><tr><td>RAW-264.7 mouse<br>macrophage reporter cell line</td><td>Laboratory of Jean-François<br>Tanti (Nice)</td><td>N/A</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>Mouse: Piezo1<sup>lox/lox</sup>/J</td><td>The Jackson laboratory</td><td>Cat#029213</td></tr><tr><td>Mouse: B6.129S7-Ldlr<sup>tm1Her</sup>/J</td><td>The Jackson laboratory</td><td>Cat#002207</td></tr><tr><td>Mouse: C57BL/6-Tg (Lysm Cre)</td><td>The Jackson laboratory</td><td>Cat#004781</td></tr><tr><td>Mouse: Piezo1<sup>lox/lox</sup>/Lysm Cre</td><td>This paper</td><td>N/A</td></tr><tr><td>Mouse: B6. Cg-Gt (ROSA)<br>26Sor<sup>tm14(CAG-tdTomato)Hze</sup>/J</td><td>The Jackson laboratory</td><td>Cat#007914</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>siPiezo1 (H1) Mm_2310061F22Rik_1</td><td>Qiagen</td><td>Cat# SI00814807</td></tr><tr><td>siPiezo1 (H2) Mm_BC039210_3</td><td>Qiagen</td><td>Cat# SI04420402</td></tr><tr><td>Negative Control siRNA (siNT)</td><td>Qiagen</td><td>Cat#1027310</td></tr><tr><td>Primers for RT-qPCR, see Table S1</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>mCherry-IRES2-mCherry</td><td>Arhatte et al., 2019<sup>64</sup></td><td>N/A</td></tr><tr><td>mPiezo1-IRES2-mCherry</td><td>This paper</td><td>N/A</td></tr><tr><td>mPiezo1-IRES2-EGFP</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>LightCycler® software release 1.5.0</td><td>Roche Life Science</td><td>N/A</td></tr><tr><td>pCLAMP10.7</td><td>Molecular Devices</td><td>https://support.moleculardevices.com/s/article/Axon-pCLAMP-10-Electrophysiology-Data-Acquisition-Analysis-Software-Download-Page</td></tr><tr><td>Fiji/ImageJ</td><td>NIH, RSB</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Vibration Free System</td><td>N-biotek</td><td>N/A</td></tr><tr><td>GraphPad Prism</td><td>GraphPad software</td><td>https://www.graphpad.com:443/scientific-software/prism/, RRID:SCR_002798</td></tr><tr><td>Metafluor software</td><td>Princeton</td><td>N/A</td></tr><tr><td>Origin software</td><td>Originlab</td><td>N/A</td></tr><tr><td colspan="3"><strong>Other</strong></td></tr><tr><td>Axopatch 700A amplifier</td><td>Molecular Devices</td><td>N/A</td></tr><tr><td>Digidata 1322A digitizer</td><td>Molecular Devices</td><td>N/A</td></tr><tr><td>PC-10 puller</td><td>Narishige</td><td>N/A</td></tr><tr><td>High Speed Pressure Clamp-1 system (HSPC)</td><td>ALA Scientific</td><td>N/A</td></tr><tr><td>LSM780 confocal fluorescence microscope</td><td>Carl Zeiss</td><td>https://www.ipmc.cnrs.fr/cgi-bin/site.cgi</td></tr><tr><td>IX83 inverted microscope</td><td>Olympus</td><td>N/A</td></tr><tr><td>FP-8300 Spectrofluorometer</td><td>JASCO</td><td>N/A</td></tr><tr><td>Videomicroscope Zeiss Axiovert200M</td><td>Carl Zeiss</td><td>https://www.ipmc.cnrs.fr/fr/facility/2imagerie-de-cytometrie/</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>AxioObserver</td><td>Carl Zeiss</td><td>N/A</td></tr><tr><td>TC-344B temperature/heater controllers</td><td>Warner Instrument Corporation</td><td>N/A</td></tr><tr><td>Lambda 721</td><td>Sutter Instrument</td><td>https://www.sutter.com/IMAGING/lambda721.html</td></tr><tr><td>EMCCD camera Evolve 512</td><td>Roper Scientific</td><td>N/A</td></tr><tr><td>Glass-bottom fluorodishes</td><td>World Precision Instruments</td><td>Cat#FD35-100</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND SUBJECT DETAILS

Piezo1 knockout mice (pure C57BL/6J background) were generated as previously described.⁶⁵ LysM Cre mice (strain 004781) were obtained from the Jackson Laboratories. For macrophage-specific Piezo1 invalidation, the LysM Cre line was crossed with Piezo1del/lox mice and with the tdTomato reporter line A14 (Strain 007914) yielding the LysM Cre Piezo1del/− A14 mice (KO). Control mice were LysM Cre Piezo1+/+ A14 (WT). The Ldlr-/- (Ldlr<sup>tm1Her/</sup>/J; strain 002207) mice were used as described below to generate an AS model when fed a Western diet. Mice were housed under a 12-h light/dark cycle with food and water provided ad libitum at the IPMC and C3M animal care facilities. All animal use and experiments were approved by our local ethical committee (CIEPAL-Azur) and carried out in accordance with EU animal care guidelines. The ages of the animals used in our experiments ranged from 8 to 14 weeks. Animals were randomly assigned to different experimental conditions. Group sample sizes were chosen on the basis of previous studies.⁶⁵,⁶⁶

## METHOD DETAILS

### AS Ldlr-/- model

#### Bone marrow transplantation

12-to-16-week-old female Ldlr-/- recipient mice were irradiated for 16 h before transplantation. Then, mice underwent i.v. injection with 4 x 10⁶ bone marrow cells from donor male mice. Transplanted mice were allowed to recover for five weeks before feeding them an atherogenic diet (Western diet, TD88137, Ssniff) for 12 weeks.

#### Analysis of atherosclerotic plaques

Mice were killed and slowly perfused with 10 mL ice-cold PBS injected into the heart. The hearts were embedded in paraffin for analysis of tissue architecture by H&E staining. Then, 10-µm paraffin sections of the aortic sinus were prepared. Histological slides were taken with a Nikon upright microscope coupled with a DS-Ri 1 color camera (Nikon Eclipse Ci). Images were acquired with a Ds-L3 software (Nikon) and the plaque area were quantified with Fiji software.

#### Blood leukocyte analysis

Leukocytes were quantified from 50 µL of whole blood collected into EDTA tubes using a hematology cell counter (Hemavet, Beckman Coulter).

#### Tissue leukocyte analysis

Cells were collected from tissues after cardiac perfusion with PBS. The aorta was cut into small pieces and digested for 30 min with HBSS medium containing 1.5 mg mL⁻¹ collagenase D (Sigma, 11088882001) and 0.5 U mL⁻¹ Liberase (Sigma, 5401054001) at 37°C. Single-cell suspensions were subjected to red blood cell lysis, filtration and centrifugation for 5 min at 400g, before incubation for 30 min on ice protected from light, with a cocktail of fluorochrome-conjugated antibodies. The antibodies used at 1:200 dilution were: CD64 (FcγRI) Brilliant Violet 768 (clone X54-5/7.1, BioLegend), CD45 APC-Cy7 (clone REA737, Miltenyi Biotec) and FOLR2 APC (clone 94b, BioLegend). Cells were then washed, centrifuged and data were acquired on Cytek Aurora flow cytometer with a five-laser configuration. Forward scatter/side scatter (FSC/SSC) gating was used to exclude dead cells and debris followed by FSC-A/FSC-H gating to select singlets. Analysis of cell subsets was performed using FlowJo software (Tree Star).

### Cell cultures, transient transfection, macrophages polarization

Bone-marrow-derived macrophages (BMDMs) were harvested from the femurs and tibias of 8-14-week-old C57BL/6 J mice. Bone marrow cells were collected by flushing the bones with ice-cold PBS. To remove red blood cells, the collected bone marrow cells were treated with red cell lysis buffer for 1 min. After sequential filtration through 70 and 40 µm Nylon filter, followed by multiple centrifugations (10 min, 500xg) cells were re-suspended in DMEM (4.5 g L⁻¹) supplemented with 10% newborn calf serum, 1% GlutaMAX, 1% Penicillin, 1% Streptomycin, and 10 ng/mL recombinant mouse macrophage colony stimulating factor (M-CSF). Growth medium was renewed at day 4. After 7 days of differentiation cells were used for further experimentation. For in vitro stimulation cells were polarized toward M1 macrophages by adding LPS (10 ng/mL) (from Escherichia coli, serotype 0111:B4) in the presence of IFN-γ (20 ng/mL) or toward M2 cells via the addition of IL-4 combined with IL-13 (20 ng/mL both) for 24 h. Unpolarized BMDMs cells (M0) were used as a control. All cell cultures were kept at 37°C in a humidified atmosphere with 5% CO₂.
The RAW-264.7 mouse macrophage cell line (RAW cells; a kind gift from Dr. J.F. Tanti, Nice) was grown in DMEM high glucose (4.5 g L⁻¹) media, supplemented with 2mM L-glutamine, 10% heat-inactivated fetal bovine serum, 1% Penicillin, 1% Streptomycin, 100 µg/mL Normocin and 200 µg/mL Zeocin. Growth medium was renewed twice a week. Cells were maintained via weekly passage by scraping and used for up to 12 passages at 60-80% confluence. Polarization toward M1 and M2 was induced as for BMDMs cells. Unpolarized RAW cells (M0) were used as a control.

Human Embryonic Kidney (HEK Piezo1<sup>del</sup>) cell line was a gift from Dr. Patapoutian's lab. Cells were maintained at a density of 2000 cells/cm² in Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g L⁻¹ glucose (Gibco BRL Life Technologies), 10% heat-inactivated fetal bovine serum (FBS; 30 min at 56°C, Hyclone), 1% Penicillin, 1% Streptomycin, and 1% Glutamax in continuous culture for up to 30 passages. Cells were dissociated with 0.05% Trypsin-EDTA and re-plated onto poly-L-lysine coated 35-mm Petri dishes. Standard molecular cloning techniques were used for plasmids and the constructs were verified by full-length sequencing. Mouse Piezo1 was cloned into an IRES2-mCherry vector. For electrophysiology, 100 000 cells were transfected with 2 µg Piezo1 using jetPEI (Polyplus) according to the manufacturer's instruction. Cells were tested 48-72 h post-transfection.

## Identification of macrophages

The proper differentiation of bone marrow progenitor cells toward macrophages was confirmed by staining with F4/80 AF488 (murine macrophage-specific surface expression marker) and visualized under fluorescence microscope. Cells were grown on collagen I coated glass bottom fluorodish, washed twice with PBS, blocked for 1 h at 4°C in buffer containing PBS, 3% BSA free-fatty acids and 2.5 mM EDTA, and stained with F4/80 monoclonal antibody conjugated to Alexa Fluor 488 (1:200) for 30 min at 4°C on orbital shaker in the dark. Live cells were counterstained with Hoechst 33342 (for nuclei) and visualized under microscope (Zeiss, Axiovert200M). For lipid droplets staining, BMDMs were incubated on collagen I coated glass bottom fluorodish, polarized to M1 in complete media (10% FBS) for 24 h, and treated with 7-KC (10 µM) for additional 24 h (LDL-free medium or 10% FBS). Cells were incubated on shaker with 80 rpm speed. Cells were washed twice with PBS, stained with BODIPY 493/503 (2 µM in PBS) for 15 min at 37°C, washed three times with PSB, and fixed with 4% formaldehyde for 20 min in room temperature (in the dark). After three times washing with PBS, cells were stained with HCS Red cell mask (2 µg/mL in PBS) for 30 min at room temperature. Cells were washed three times with PBS and stain with DAPI (1 µg/mL) for 10 min, at room temperature. Images were taken with a Zeiss LSM780 confocal microscope.

## Pharmacological treatments

LDL and oxLDL were purchased from Thermo Fisher Scientific. Cells were treated with 50 µg/mL of compound in LDL-depleted media (10% LDL-free serum) and cultured for 24 h.

7-ketocholesterol (7-KC) was obtained from Sigma-Aldrich. The stock solution was prepared by dissolving the chemical compound in 100% ethanol at the concentration of 50 mM and bath sonicated for 10 min. Cells were treated with 7-KC in LDL-depleted media (10% LDL-free serum) and cultured for 24 h. For HEK Piezo1<sup>del</sup>, 7-KC was added 48 h post-transfection and cells were incubated for additional 24 h. Control cells were incubated with the corresponding vehicle (EtOH). No sign of cell death (LDH release) was detected at a concentration of 10 µM or below, whereas deleterious effects were visible at 25 µM (24 h treatment in LDL-free conditions) (not shown). A 7-KC concentration of about 0.06 µg/g tissue was estimated in the aorta of Ldl<sup>+/-</sup> mice fed a high cholesterol diet.<sup>67</sup> Moreover, circulating lipoprotein 7-KC concentration is about 80 µg/mL (i.e., 200 µM) and 7-KC mass in macrophages 200 nmol/mg in mice fed a Western diet.<sup>68</sup> Thus, a chronic treatment with 10 µM 7-KC was considered to be within the pathophysiological range. DHA (Sigma-Aldrich) was dissolved in ethanol at a concentration of 100 mM and kept at -20°C under argon. DHA was tested at a concentration of 5 and 10 µM, versus EtOH.

## Preparation of lipoprotein-depleted fetal bovine serum (LDL-free serum)

Lipoprotein depletion of fetal bovine serum (FBS) was achieved by density gradient ultracentrifugation.<sup>69</sup> The initial density of FBS (1.006 g/mL) was adjusted to 1.215 g/mL by adding potassium bromide (KBr), based on the Radding-Steinberg equation: X = ([V(d<sub>f</sub> - d<sub>i</sub>)])/([1 - (0.312 × d<sub>i</sub>)]), where: X = weight of KBr in grams, V = volume of FBS in ml, d<sub>f</sub> = final density of FBS (1.215 g/mL), d<sub>i</sub> = initial density of FBS (1.006 g/mL), 0.312 = partial specific volume of KBr. Density-adjusted FBS was centrifuged at 48,000 rpm (70 Ti rotor, Beckman Coulter) for 48 h at 4°C. The top layer containing lipoproteins was removed, and the bottom layer was collected and dialyzed against a buffer containing 150 mM NaCl and 0.3 mM Na<sub>2</sub>EDTA for 68 h (five buffer changes). After dialysis, the serum was filter sterilized and stored at -20°C.

## siRNAs transfection

For siRNA experiments, 20 nM total siRNA was transfected by using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's instruction. RNA samples were isolated from cells 72 h post-transfection. The following siRNAs were used for RAW transfections: mouse PIEZO1 siRNA-1 (Mm_2310061F22Rik_1, Qiagen), mouse PIEZO1 siRNA-2 (Mm_BC039210_3, Qiagen), and Non-targeting siRNA (Qiagen) as a negative control. siRNAs were previously validated elsewhere.<sup>47</sup>
Electrophysiology

Cells were plated on 35 mm non- or poly-L-lysine coated plastic dishes (RAW/BMDMs and HEK Piezo1<sup>del</sup>, respectively). HEK cells were recorded 48–72 h post-transfection. Data were acquired using an Axopatch 700A amplifier (Axon Instruments), Digidata 1322A digitizer (Molecular Devices) and pCLAMP10.7 software. Currents were sampled at 20 kHz and filtered at 1 kHz. All patch clamp recordings were performed in standard (cell-attached), or excised (inside-out, outside-out) patch configurations at room temperature. Patch pipettes were made from 1.5 mm outer diameter glass (Vitrex Medical, #160214) using a Narishige PC-10 puller, and had resistance of 1.2–1.4 MΩ when filled with a saline solution consisting of (in mM) 150 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES (pH adjusted to 7.35 with NaOH)(External buffer). The bath solution consisted of (in mM) 155 KCl, 3 MgCl₂, 5 EGTA, 10 HEPES (pH adjusted to 7.2 with KOH).<sup>47,65</sup> The osmolality of all solutions was 310 mOsmol.kg<sup>-1</sup>. Membrane patches were subjected to stepwise, 350-ms long pressure pulses (A: 10 mm Hg) through the recording electrode using a fast pressure-clamp device (High Speed Pressure Clamp-1 system, ALA-scientific). Pressure effect curves were fitted with a Boltzmann function: I(P) = [1 + exp(-(P - P<sub>50</sub>)/k)]<sup>-1</sup>, where I is the peak of stretch-activated current at a given pressure, P is the applied patch pressure (in mm Hg), P<sub>50</sub> is the pressure value that evoked a current value which is 50% of Imax, and k the slope. Time to peak current was determined between the onset of pressure pulse and the maximal current amplitude. Inactivation kinetics of mechano-evoked currents were determined by fitting current decay to a single exponential decay function: I = ΔIexp(-t/τ), where ΔI is the difference between peak MA current and baseline, t is the time from the peak current (the start of the fit), and τ is the decay constant (HEK Piezo1<sup>del</sup>). For BMDMs and RAW cells Ipeak/Isteady ratios (maximal current amplitude/amplitude at the end of pressure pulse) were measured. Deactivation kinetic was either determined as a mono-exponential fit (HEK Piezo1<sup>del</sup>) of the tail current or measured at 1 s post pressure pulse. Yoda1 was dissolved at 50 mM in DMSO and used at a final concentration of 5 µM. DHA was dissolved in ethanol at 100 mM and stored at -20°C under argon and tested at 5 and 10 µM. Both drugs were applied by superfusion in the medium and the vehicles (DMSO or ethanol) were added in the control solutions.

Calcium imaging

BMDMs (M0) were plated on glass-bottom fluorodishes (World Precision Instruments) coated with collagen I. Cells were loaded with 2 µM Fura-2 AM (Molecular Probes) for 30 min and visualized under an inverted epi-fluorescence microscope (AxioObserver, Carl Zeiss, France) using a Fluar x20. Then cells were superfused with control (DMSO) or Yoda1 (10 µM) solutions at 37°C using an automatic heater controller system (TC-344B temperature/heater controllers, Warner Instrument Corporation, Hamden, USA). The control solution contained (mmol/L): NaCl, 132; KCl, 5.6; CaCl₂, 1.8; MgCl₂, 1.2; NaHCO₃, 5; NaH₂PO₄, 1; HEPES, 20; glucose 1 g/L, pH 7.35. Illumination was performed with a Sutter Instrument Lambda 721. Fura-2 was sequentially excited at 340 nm and 380 nm, and the emission monitored at 510 nm. Images were acquired with a cascade 512 EMCCD camera (Cascade 512, Roper Scientific, Evry, France) every 2 s using the Metafluor software (Metafluor, Princeton, NJ, USA). 340 and 380 nm background fluorescence signals were collected at the same rate and subsequently subtracted from respective fluorescent images. Cells with increased ΔR/R0 in response to Yoda1 (10 µM) were counted from fields taken from different experiments. A Ca²⁺-free solution was made by omitting calcium and adding 100 µM EGTA. For calibration of calcium concentration, we used the Ca²⁺-free solution supplemented with 5 µM ionomycin for determination of the minimal ratio (Rmin) and the control solution supplemented with 8 mM calcium and 5 µM ionomycin for the maximal ratio (Rmax). Final free calcium concentration was calculated using a macro under Origin software (Originlab, Northampton). Intracellular calcium values (in nm) were calculated according to the equation: [Ca] = Kd * (R-Rmin)/(Rmax - R) * Sf2/Sb2. The values of Rmax and Rmin are the ratio values measured under conditions of saturating calcium levels and in the absence of calcium respectively. The values of Sb2 and Sf2 are proportional to the fluorescence excited by the denominator wavelength (380 nm) again under conditions of saturating calcium levels (the “b” referring to the bound state) and in the absence of calcium (the “f” referring to calcium free) respectively. A value of 140 nM was used for the dissociation constant for the Fura-2-Calcium binding (Kd).

Real time qPCR

RAW cells were harvested 72 h post siRNA transfection, and BMDMs 24 h post 7-KC treatment. Total RNA was extracted using RNeasy MINI KIT (Qiagen) and equal amounts of cDNA were synthesized using GoScript reverse transcriptase (Promega). qPCR experiments, in duplicate from minimum three independent transfections, or minimum two WT/KO mice, were performed using SybrGreen (Eurogentec). The data were normalized to the mouse topoisomerase 1 (TOP1) reference gene expression. Oligonucleotide sequences are listed in Table S1 qPCR data were analyzed using the Light Cycler 480 software release 1.5.0 and GraphPad Prism program.

Confocal imaging of Piezo1 membrane expression

HEK Piezo1<sup>del</sup> cells were plated on 35-mm collagen I coated fluorodish and co-transfected with 2 µg Piezo1-EGFP (Ct fusion) plasmid (for fluorescence membrane quantification) together with 0.2 µg mCherry-IRES2-mCherry (for cytosolic mask).<sup>47</sup> 48 h post-transfection cells were treated for 24 h with 10 µM 7-KC (or EtOH vehicle) in LDL-free media. Piezo1-EGFP and mCherry cytosolic stainings were imaged on living cells 72 h after transfection by laser scanning confocal fluorescence microscopy using a 63x/1.4 objective (LSM780, Carl Zeiss, Rueil Malmaison, France). The quantitative analysis of Piezo1 expression at the plasma membrane was done using the ImageJ/Fiji software (Schindelin et al., 2012). A mask based on the cytosolic area (mCherry signal) was applied for each cell to specifically measure plasma membrane Piezo1 fluorescence.
## Piezo1 immunofluorescence

BMDMs (M1) were treated with 7-KC (10 µM)/EtOH for 24 h (LDL-depleted medium). Cells were washed with PBS and fixed using formaldehyde (4%) in PBS for 5 min at room temperature (RT). Cells were washed again and blocked with PBS containing 1% horse serum (PBS-HS) for 30 min at RT. This was followed by incubating with Piezo1 primary antibody (1:250 dilution, Proteintech, 15939-1-AP) in PBS-HS for 2 h at 37°C. Cells were washed again and incubated with Alexa 488-tagged secondary antibody (1:1000) in PBS-HS for 1 h at 37°C. In control experiments only the secondary antibody was included. After the incubation, cells were washed and stained with DAPI (1 µg/mL) and WGA (5 µg/mL) in PBS for 10 min at RT. Cells were washed again and imaged by confocal microscopy.

## Confocal imaging and calculation of generalized polarization

Cells (HEK/RAW/BMDMs) were treated with EtOH or 7-KC (10 µM) in culture media (with LDL-depleted serum) for 24 h. After the incubation, cells were washed twice with external buffer (as described above for patch clamp experiments, added with 1 g/L glucose) and incubated with NR12S (40 nM) at 37°C for 10 min. Cells were imaged using a confocal microscope (Zeiss, LSM780). Samples were excited at 488 nm laser, and the fluorescence emission was collected from 550 to 600 nm (ordered channel, IO) and 600-650 nm (disordered channel, ID).⁷⁰ Generalized polarization (GP) values were calculated based on the formula $GP = (IO-ID)/(IO + ID)$ using an in-house ImageJ plug-in.

## DHE/MβCD complex preparation

DHE (5 mM) stock was prepared in ethanol, dried under vacuum and mixed with MβCD (25 mM in external buffer, as described above for patch clamp experiments) to obtain DHE:MβCD ratio of 1:5.⁷¹ This mixture was bath sonicated for 10 min and incubated overnight at 37°C. The next day, clear supernatant was collected by centrifuging at 20,000g for 10 min. DHE:MβCD aliquots were stored at 4°C under argon, and protected from light. In control experiments, we initially verified that 7-KC (10 µM) does not cause a quenching of 10 µM DHE fluorescence when dissolved either in pure EtOH or in a saline solution (DHE imaging buffer). In organic solvents, DHE shows an excitation spectrum with three maxima at 311, 324, and 340 nm and an emission spectrum with three maxima at 354, 371, and 390 nm. The emission maxima shift to 404 and 426 nm in aqueous conditions. DHE (10 µM) solution (in ethanol or buffer) was excited at 324 nm and collected the fluorescence emission spectrum from 350 to 500 nm using a fluorometer (JASCO FP-8300) at room temperature. DHE solution was then mixed with 7-KC (10 µM) and fluorescence measurement repeated. Fluorescence spectra from 7-KC (in ethanol or buffer) and the solvents were also collected as background controls. In ethanol, DHE showed a more intense fluorescence, as compared to saline buffer. Notably, in both conditions, 7-KC incubation did not change the fluorescence intensity or the spectrum of DHE. This shows that 7-KC does not quench the DHE fluorescence in either organic or aqueous environments.

## DHE labeling protocol

HEK Piezo1<sup>del</sup> or RAW cells (0.05 million/mL, 700 µL) were seeded on collagen I coated 4 well plates (Ibidi) and incubated for 24 h for the attachment on the surface. For RAW cells, M1 or M2 polarization was induced for 24 h as described before. HEK Piezo1<sup>del</sup> cells were transfected with mCherry-IRES2-mCherry (0.4 µg of plasmid per plate) or Piezo1-IRES2-mCherry plasmid (1 µg of plasmid per plate) for 48 h. For DHE labeling, cells were washed thrice in the external solution followed by incubation with 50 µL of DHE:MβCD complex for 2 min at 37°C. Cells were washed thrice and incubated further at 37°C for 15 min to allow the uniform distribution of DHE across the cells. Cells were next incubated with LDL-free culture medium (10% LDL-depleted serum) containing 7-KC (10 µM) or EtOH for 8 h. Cells were shaken at 80 rpm using an orbital shaker to provide mechanical stimulation by shear stress. After incubation, HEK Piezo1<sup>del</sup> cells were washed and imaged using a fluorescence microscope. RAW cells were counter-stained with WGA (5 µg/mL) for 15 min at room temperature, followed by washing (3 times) and imaged using an inverted fluorescence microscope (Olympus, IX83).⁷² Live cells were kept in the external solution containing glucose (1 g/L) during imaging. Images were analyzed using an in-house ImageJ plug-in to measure the mean membrane fluorescence.

## High performance thin-layer chromatography (HPTLC)

### Reagents and standards for lipid analysis

Lipid standards (reference, abbreviation) were acquired from Sigma and Avanti Polar Lipids: L-α-lyso-phosphatidylcholine (830071, LPC), sphingomyelin (860062, SM), L-α-phosphatidylcholine (840051, PC), L-α-phosphatidylserine (840032, PS), L-α-phosphatidylinositol (840042, PI), cardiolipin (840012, CL), L-α-phosphatidylethanolamine (840026, PE), L-α-phosphatidylglycerol (841138, PG), C24 Lactosyl(β) Ceramide (d18:1/24:0) (860577, Hex2Cer), Mono-Sulfo Galactosyl(β) Ceramide (d18:1/17:0) (860572, MSGCer), Glucosyl(β) Ceramide (d18:1/17:0) (860569, HexCer), ceramide (860051, Cer), 1-oleoyl-rac-glycerol (330724, MG), 5-cholestén-3β-ol (C8667, ST), cholest-5-en-3β-yl heptadecanoate (700186, STE), cholesteryl formate (S448532, ChoF), arachidonic acid (A3611, FFA), 1,3-di(cis-9-octadecenoyl)-glycerol (D3627, DG1.3), 1,2-dipalmitoyl-sn-glycerol (800816, DG1.2), 1,2,3-tri-(9Z-octadecenoyl)-glycerol (T7140, TG).

Chloroform (CHCl3), methanol (MeOH) and hexane were HPLC grade; toluene (Sigma, 244511); ethyl acetate (VWR, 23880.290, EtAc); 1-propanol (Sigma, 402893). Other reagents: butylhydroxytoluene (Sigma, 34750, BHT), ammonium molybdate tetrahydrate (Sigma, A7302), ascorbic acid (Sigma, 33034), sodium dihydrogen phosphate dihydrate (28011.291), perchloric acid 65-71% (Carlo
Erba, 409193), nitric acid 65% (VWR Chemicals, 20429.245), sulfuric acid 95% (VWR Chemicals, 20700.298), phosphoric acid (Sigma, 1.00565), copper (II) sulfate pentahydrate (Sigma, C8027).

## Lipid extraction and quantification

Samples were washed in PBS, pelleted and stored frozen. After thawing, they were transferred into glass tubes and extracted with a modified Bligh & Dyer method⁷³: volumes were scaled down and a re-extraction step was added (clean organic phase into the remaining aqueous phase), after which the two collected organic phases were combined. All extraction solvents had 50 µg/mL BHT. Extracted samples were kept at -20°C under argon.

Phospholipid quantification was performed according to,⁷⁴ with scaled down volumes. Standards were prepared with sodium di-hydrogen phosphate dihydrate (0.1–1 µg P). Phospholipid mass was calculated based on an average MW of 775 g/mol.

## HPTLC

An automatic TLC sampler 4 (ATS4, CAMAG) with a 25 µL spray needle was used to apply samples (20 µg PL) and standards (5 µg) onto a Merck HPTLC glass plate silica gel 60 (20 × 10 cm, layer thickness 200 µm), at Y = 8 mm, X = 20 mm, and band length, width and distance of 6, 0, 9 mm, respectively. Dosage speed was 110 nL/s. The syringe was washed 3x between each application with CHCl₃:MeOH (1:1). The plate was then eluted with 8 different solvent mixes as described elsewhere (https://doi.org/10.2139/ssrn.4951623). Plate surface was sprayed (red nozzle, speed 4) with a modified copper sulfate solution (https://doi.org/10.1080/10826070802188940) in a derivatization chamber (CAMAG) and revealed by heating at 100° C in a plate heater (CAMAG) inside a fume hood for 90 min, then 5 min at 140° C. Imaging was done with a Fusion FX7 instrument (Vilber Loumat), using epi white light. Images were treated in Fiji. Chromatograms were obtained after background removal, and plotted using GraphPad Prism v10.

## QUANTIFICATION AND STATISTICAL ANALYSIS

When appropriate, significance of the differences was tested using the GraphPad Prism software. The sample sizes were chosen based on common practice in physiology and patch clamp experiments. No statistical methods were used to predetermine sample size. Statistical tests were chosen based on normality of Gaussian distributions determined with a D'Agostino & Pearson omnibus normality test. If data met normality a parametric test was used (two samples Student's t test). If not, a nonparametric test was used (Mann-Whitney U test). For groups with small *n* values (*n* < 30), in which normality cannot be accurately established, a nonparametric test was used. One star indicates *p* < 0.05, two stars *p* < 0.01, three stars *p* < 0.001 and four stars *p* < 0.0001. Significance was defined as a *p* value of 0.05 or less. Data represent mean ± standard error of the mean (SEM). Regression analysis was performed using Boltzmann sigmoidal distribution. The number of independent times each experiment has been repeated (n) is indicated throughout the manuscript and shown in figures for mean currents. N indicates the number of mice.