Role of Piezo1 in pulmonary arterial hypertension – HTAP

Since constitutive Piezo1-deficient mice die during early development, a conditional SMC specific Piezo1 KO mouse line was engineered (team 1), giving us a unique opportunity to study the physiological role of Piezo1/MSCs in adult mice . We use either a Piezo1lox/lox or Piezo1 del/lox (one allele is constitutively deleted, thus allowing an optimal invalidation of Piezo1 upon Cre activation and deletion of the second Piezo1 floxed allele) mouse model to invalidate Piezo1 specifically in SMCs. Piezo1 currents are recorded using the cell-attached patch clamp configuration (Team 1).
After 6 weeks of tamoxifen induction, both WT and KO mice (15 to 20 weeks old) are kept in either normoxic or hypoxic conditions (exposure to 9% oxygen for 21 days) (team 2). PH will is assessed based on determination of the right ventricular (RV) pressure and assessment of RV hypertrophy (Fulton index). Moreover, lung sections are labeled with sm22 and PECAM-1 antibodies to visualize SMCs and endothelial cells. Cell proliferation is detected with Ki67 and PCNA antibodies. Fibrosis is visualized with antibodies against collagens, as well as Picrosirius red staining. A morphometric analysis of intrapulmonary arteries is further performed (inner diameter, wall thickness, cross-sectional area and media/lumen ratio).

1. Relationship between Piezo1 and mechanosensitive ion channels in pulmonary artery smooth muscle

We generated a conditional invalidation (knock-out; KO) of Piezo1 specifically in smooth muscle cells (SMCs). We isolated SMCs from pulmonary arteries (PAs) of TAM-injected Piezo1lox/lox (control; WT) and Piezo1del/lox (KO) mice. We recorded ion channel activity in response to a localized increase in pressure applied at the patch clamp pipette. Our results demonstrate the existence of high-amplitude inward currents in response to negative pressure in arterial SMCs with a reversal potential of 0 mV. Notably, these currents are characterized by a very slow inactivation. The mechanosensitive current amplitude is significantly reduced in Piezo1 KO cells.

This first part of the project allowed us to quantify the expression of Piezo1 in mouse PA SMCs. In addition, we established the functional link between Piezo1 and mechanosensitive ion channels in mouse PA SMCs.

2. Role of Piezo1 in experimental pulmonary hypertension in mice

Mice (WT and KO) were subjected to chronic hypoxia (9% oxygen) for 21 days compared with normoxia (21% oxygen). We observed that Piezo1 invalidation (either using a Piezo1 lox/lox or Piezo1 del/lox genotype) significantly reduced RVSP and Fulton index in chronically hypoxic mice. In addition, histological analysis indicates that arterial remodeling caused by chronic hypoxia was also significantly reduced upon conditional invalidation of Piezo1 in SMCs.

These results established for the first time the functional role of Piezo1 in SMCs in the remodeling of mouse PAs in response to chronic hypoxia. Taken together, these results demonstrate an important role for mechanical stress and Piezo1 in the etiology of pulmonary hypertension (PH).


3. Role of Piezo1 in hypoxic pulmonary vasoconstriction

A characteristic feature of the pulmonary circulation is its ability to contract in response to acute hypoxia (hypoxic pulmonary vasoconstriction or HPV). In complementary experiments, we induced HPV in vivo by ventilating mice with a hypoxic gas mixture (9% hypoxia) for 5 minutes. The increase in RVSP caused by acute hypoxia was significantly decreased following Piezo1 invalidation in the SMCs. These results were confirmed in vitro using a perfused isolated lung model (both rapid and delayed components of HPV are inhibited following Piezo1 invalidation in SMCs, in contrast to the vasoconstriction induced by U46619.

Overall, our results show that Piezo1 in SMCs is involved in PA responses to acute hypoxia (HPV) and secondarily influences arterial remodeling during PH (chronic hypoxia).

In summary, during this project we established the link between Piezo1 and the mechanosensitive ion channels of PA myocytes. Furthermore, we identified the role of Piezo1 in HPV and PH. Finally, we showed that pharmacological inhibition of Piezo1 reduces the proliferation of PA SMCs from PH patients.

Our studies of molecular and integrative physiology will allow the interrogation of the role of Piezo1 in PH, using both mouse and human pathologic tissues. Our main hypothesis relies on preliminary findings showing that SMCs specific conditional genetic invalidation of Piezo1 in the mouse is protective against chronic hypoxia-induced PH and reduces HPV. These findings will shed new light on the molecular mechanisms of PH and will open up new avenues for possible therapeutic strategies. In this line, we will explore whether or not inhibition of Piezo1 by chronic dietary margaric acid (MA) may confer a protection in vivo against experimental PH in the mouse and in vitro against the hyperproliferation of PA SMCs from PH patients. Thus, the expected impact of the project will be academic (beneficial for the fields of mechanobiology, ion channels and vascular physiology), but also hopefully with possible translational applications (beneficial for the society) for the fight against PH, that remains a devastating incurable disease.

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In pulmonary arterial hypertension (PH), both an increase in arterial reactivity and a structural remodeling, causing a thickening of the muscular medial layer of small pulmonary arteries, are implicated in the etiology of the disease. A better understanding of the molecular and cellular basis of PH is urgently needed to identify possible therapeutic options. Several arguments point to an important role for mechanical stress in the stimulation of pulmonary artery smooth muscle cells proliferation. We hypothesize that opening of the mechanosensitive ion channel Piezo1 in pulmonary artery myocytes is implicated in PH.
Our specific aims are: 1) to establish a functional link between Piezo1 and the mechanosensitive ion channels in pulmonary artery smooth muscle cells; 2) to identify the role of Piezo1 in hypoxic pulmonary vasoconstriction and pulmonary artery remodeling associated with chronic hypoxia; 3) to explore the signaling pathways downstream of Piezo1 in pulmonary artery myocytes; 4) to examine whether or not the pharmacological inhibition of Piezo1 confers a protection against PH.