Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells, which act collectively through robust cell–cell interactions, take on mesenchymal characteristics, breaking cell–cell junctions to become solitary, invasive and motile. Our previous results show that a transient increase in calcium influxes through TRP channels at the plasma membrane is required for hepatocyte growth factor (HGF)– stimulated EMT. Since this transient increase requires an intact microtubule cytoskeleton, we propose that HGF stimulation results in the mobilization of calcium channels to the plasma membrane from an intracellular compartment via microtubule–dependent vesicle trafficking. Through immunofluorescence, we show that prior to HGF treatment, TRPV4 localizes to a perinuclear compartment that stains for rab11. After HGF stimulation, this colocalization is reduced and TRPV4 localizes more precisely to fibrous structures. Similarly, rab8 staining is seen throughout the cytoplasm prior to HGF treatment, but localizes primarily to tubular structures after HGF stimulation. This is indicative of endocytic recycling of TRPV4 via rab8. MDCK cells null for rab8 activator, rabin8, were developed using the CRISPR system and then analyzed for changes in epithelial scattering and trafficking of ion channels to the plasma membrane following HGF stimulation. Rabin8 KO cells had a decrease in TRPV4 vesicle trafficking. While rabin8 KO cells did undergo HGF-induced spreading and some disassembly of cell-cell junctions, they lost all motility. Also, HGF-treated rabin8 KO cells had similar calcium levels to untreated WT cells, which had fewer calcium spikes than HGF-treated WT cells. ERK1/2, a known downstream effector of HGF stimulation, has been shown to activate rabin8, and so we tested the effect of an ERK1/2 inhibitor on HGF-induced WT cells as well. These cells had decreased TRPV4 vesicle trafficking and loss of motility, similar to rabin8 KO cells, indicating that ERK1/2 may act upstream of rabin8 and rab8 in this pathway. Our results indicate that TRPV4 undergoes endocytic recycling via rab8 to the cell surface to allow a necessary calcium influx within one hour of HGF stimulation in MDCK cells, leading to EMT.

Transient receptor potential (TRP) channels play an important role in sensory and nonsensory functions. TRPVanilloid 1 and TRPVanilloid 4 are proposed to be involved in inflammation-induced pain. TRPV1 is extensively studied and it is specifically involved in inflammatory thermal hypersensitivity. Mechanical hypersensitivity is one of the significant components of nociception. Several receptors have been proposed to underlie mechanosensation. The molecular entities responsible for mechanosensation are not fully understood. In this study, I have characterized the properties of TRPV4, a putative mechanosensitive ion channel expressed in dorsal root ganglion (DRG) neurons and nonsensory tissues. First, I have investigated the expression and function of TRPV4 and TRPV1 in the DRG neuronal cell bodies as well as their central terminals and determined the modulation by protein kinase C (PKC). Both TRPV4 and TRPV1 are expressed in DRG and laminae I and II of the spinal dorsal horn (DH). Ca2+ fluorescence imaging and whole-cell patch-clamp experiments showed that both capsaicin-induced TRPV1 response and 4alpha-phorbol 12, 13-didecanoate (4alpha-PDD)-induced TRPV4 response were observed in a proportion of the same DRG neurons, suggesting their co-expression. Incubation of DRG neurons with phorbol 12, 13-dibutyrate (PDBu), a PKC activator, resulted in a significantly greater potentiation of TRPV4 currents than TRPV1 currents. In HEK cells heterologously expressing TRPV4, PDBu potentiated TRPV4-mediated single-channel current activity. In patch-clamped DH neurons, the application of 4alpha-PDD at the first sensory synapse increased the frequency but not the amplitude of the miniature excitatory postsynaptic currents (mEPSCs), suggesting a presynaptic locus of action. 4alpha-PDD-induced increase in the frequency of mEPSC was further facilitated by PDBu. These results suggest that TRPV4 in the central terminals modulates synaptic transmission and is regulated by PKC. Second, I have studied the mechanosensitivity of TRPV4 in cell-attached patches by applying direct mechanical force via the patch pipette. In TRPV4 expressing HEK cells, the application of negative pressure evoked single-channel current activity in a reversible manner and the channel activity was enhanced after incubation with PDBu. TRPV4 has been shown to be activated by hypotonicity. Here I show that negative pressure exaggerated hypotonicity-induced single-channel current activity. However, in similar experimental conditions, cells expressing TRPV1 did not respond to mechanical force. TRP channels are also expressed in non-sensory regions and the role of these channels is not fully understood. Both TRPV4 and TRPV1 are expressed in the hippocampus. Using whole-cell patch-clamp techniques, I have found that 4alpha-PDD increased the frequency, but not the amplitude of mEPSCs in cultured hippocampal neurons, suggesting a presynaptic site of action. Interestingly, the application of capsaicin had no effect on synaptic transmission in hippocampal neuronal cultures. Finally, I have investigated the expression and function of TRP channels in diabetes because TRP channels have been shown to be involved in peripheral neuropathy as well as vascular complications in diabetes. ROS production plays a critical role in the progress of diabetes. I propose that lower levels of ROS up-regulate the expression TRP channels in the early stages of diabetes, leading to hyperalgesia, and higher levels of ROS or chronic exposure to ROS down-regulate TRP channels in the late stages of diabetes, resulting in hypoalgesia. I have found that the expression of TRPV1 and phospho p38 (p-p38) MAPK was increased in DRG of streptozotocin (STZ)-injected diabetic and non-diabetic hyperalgesic mice. An increase in TRPV1 and p-p38 MAPK levels was induced by STZ or H2O2 treatment in stably TRPV1 expressing HEK cells, suggesting the involvement of STZ-ROS-p38MAPK pathway. TRPV4 has been reported to be involved in vasodilatation by shear stress in blood vessels. Here, I have demonstrated that TRPV4 is expressed in lymphatic endothelial cells (LECs). Treatment with low concentration of H2O2 enhanced the expression of TRPV4 at mRNA and protein levels in LECs, suggesting that mild levels of ROS up-regulate TRPV4 expression. In diabetes, beta cell dysfunction is responsible for decreased insulin release. TRPV4 is expressed in RINm5F (beta cell line), islets and pancreas. It has been shown that hypotonicity induced insulin release in beta cell lines, which was mediated by activation of stretch-activated channels, raising the possibility of the involvement of TRPV4, a mechanosensitive channel. Therefore, I have studied the functional role of TRPV4 in beta cells. Incubation with 4alpha-PDD enhanced insulin release in RINm5F cells, suggesting TRPV4 regulates insulin secretion from pancreatic beta cells. Since TRPV4 expression levels are decreased in diabetes, insulin secretion from beta cells may be impaired. In summary, TRPV1, a thermosensitive channel, and TRPV4, a mechanosensitive channel, contribute to thermal and mechanical hyperalgesia, respectively in the early stage of DPN through their up-regulation by ROS-p38 MAPK and insulin/IGF-1 pathways. Due to the mechanical sensitivity of TRPV4 channel, the up-regulation in the early stage and down-regulation in the late stage may be involved in the development of vascular complications and regulation of insulin release in diabetes.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a disease characterized by an increase in cerebrospinal fluid (CSF) in the ventricles of the brain. This manifests as a result of either overproduction or underabsorption of CSF leading to increases in pressure, swelling and loss of brain matter. Current treatments for this disease include surgical interventions via the introduction of shunts or endoscopic third ventriculostomy, both of which aim to redirect flow of CSF in to another cavity for absorption. Limited pharmacotherapies are available in the treatment of hydrocephalus, and there exists a clinical need for drug therapies, which can ameliorate the pathophysiology associated with hydrocephalus and ventriculomegaly. CSF is produced primarily by the choroid plexus (CP), found in the ventricles of the brain. Composed of a high resistance epithelium surrounding a capillary network, the CP epithelium acts as a barrier, regulating ion transport between the CSF and blood. Transient Receptor Potential Vanilloid-4 (TRPV4) is a nonselective Ca2+-permeable cation channel expressed in the CP which is being investigated for its role in CSF production. To study hydrocephalus, we utilize two model systems; the TMEM67-/- Wpk rat, and the PCP-R cell line. The Wpk rat model is used to study the effects of drug intervention on the development and progression of hydrocephalus. The PCP-R cell line is utilized for studies which aim to understand the mechanisms by which CSF is produced. Using Ussing chamber electrophysiology, we are able to study the role of specific channels, transporters and modulators in driving epithelial ion flux across the CP. This research aims to establish a role for TRPV4 in production and regulation of CSF, and to interrogate a mechanism by which this ion transport occurs. The chapters that follow describe components of the pathway by which TRPV4 is activated and ion flux is stimulated.

Les forces mécaniques dirigeant la valvulogénèse sont mal connues. Chez le poisson zèbre, le flux induit l’expression du facteur de transcription klf2a de façon endothéliale spécifique afin d’initier ce processus. Nous nous sommes intéressés aux mécanismes moléculaires activés par le flux et aboutissant à la formation des valves cardiaques. En altérant et modifiant le pattern fluidique, nous avons observé le rôle des forces engendrées par le flux pour le contrôle de l’expression de klf2a ainsi que du nombre de cellules endocardiques. Nous avons ensuite regardé les divers mécanosenseurs pouvant intervenir lors de ce processus. Ainsi nous avons mis à jour la présence de cils dans l’endocarde et fait la relation entre les canaux membranaires TRPP2 et TRPV4, la présence de calcium intracellulaire dans les cellules endocardiques et la voie moléculaire PKC-PKD2-HDAC5, composants nécessaires à l’expression du gène klf2a en réponse au flux. Ces données nous permettent de suggérer le rôle de TRPP2, TRPV4 et la voie moléculaire récemment découverte, PKC-PKD2-HDAC5 dans la formation valvulaire dépendante de klf2a. / Mechanical forces that dictate valve formation are not well understood. In zebrafish, blood flow induces expression of the transcription factor klf2a in a specific subset of endocardial cells that will go on to form functional valves. We aimed to identify the molecular mechanisms activating this flow-induced valve formation. By altering and modifying blood flow patterns, we observed that flow-mediated forces are necessary to control early klf2a expression and endocardial cell numbers. We then looked at different mechanosensors operating at early stages of valve development. Using in vivo labelling, we identified primary cilia in the endocardium and showed that the membrane channels TRPP2 and TRPV4 increase intracellular calcium which activates a PKC-PKD2-HDAC5 pathway necessary for klf2a expression in response to flow. Together these data suggest a role for TRPV4, TRPP2 and the recently described PKC-PKD2-HDAC5 signalling pathway in klf2a-mediated valvulogenesis.

La mucoviscidose est une maladie génétique dont l’atteinte respiratoire est responsable de 90 % de la morbidité et de la mortalité et est caractérisée par une infection chronique et une inflammation persistante. Cette inflammation non contrôlée participe de manière importante à la dégradation du tissu pulmonaire. Malgré les progrès récents, les thérapies actuelles ne permettent pas un traitement efficace de l’atteinte respiratoire. Il est donc indispensable d’identifier de nouveaux mécanismes moléculaires et cellulaires impliqués dans l’inflammation pulmonaire. Dans ce but, nous nous sommes intéressés au canal calcique "Transient Receptor Potential Vanilloid 4" (TRPV4) exprimé au niveau de l’épithélium respiratoire. A l’aide d’approches in vitro et in vivo, nous avons démontré que l’activation du TRPV4 déclenche la sécrétion de médiateurs inflammatoires cytokiniques et lipidiques et un recrutement leucocytaire dans les poumons. Nous avons également observé une altération de la signalisation dépendante du TRPV4 dans le contexte de la mucoviscidose, suggérant que le TRPV4 pourrait constituer une cible prometteuse pour le développement de nouvelles thérapies anti-inflammatoires applicables en santé respiratoire. / Cystic fibrosis (CF) is due to mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). The pulmonary consequence of the disease accunts for over 90 % of the morbidity and mortality and is characterized by chronic infection and persistent inflammation. This uncontrolled inflammation participates significantly to the degradation of the lung tissue. Despite recent progress, current therapies do not allow effecgive treatment of CF lung disease. It is therefore necessary to characterize nex cellular and molecular mechanisms that could contribute to lung inflammation. In that purpose, we focused on the calcium channel "Transient Receptor Potential Vanilloid 4" (TRPV4) expressed by respiratory epithelium. Using in vitro and in vivo approaches, we found that TRP4 activation triggers the secretion of inflammatory mediators (including cytokines and lipids) and leukocytes recruitment into the lungs. We also observed a significant alteration of TRPV4-dependent signalling in the CF context, suggesting that TRPV4 could constitue a promising target for the development of new anti-inflammatory therapies in lung diseases such as CF.

This dissertation will present the four manuscripts I published or am ready to publish on the study of the pathophysiology of migraine headache. The first chapter will discuss the background of the current understanding of migraine pathophysiology. Chapter 2 is focused on studying how Transient receptor potential vanilloid 4 (TRPV4) might play a role in migraine headache. Chapter 3 is the study of a novel cell type: dural fibroblasts might also play an active role in migraine headache. Chapter 4 is discussing Norepinephrine's role in headache pathophysiology. Chapter 5 is studying the combined effect of Acid and ATP in the pathophysiology of migraine headache. The dissertation will end in a conclusion in Chapter 6.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear. Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative in vitro model is critical to elucidate the signaling pathways orchestrating CSF production in the CP. This research aims to characterize an in vitro culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a medical condition characterized by a buildup of cerebrospinal fluid which causes hydrostatic pressure to increase resulting neuronal destruction and can ultimately cause death. Hydrocephalus is seen in both the pediatric population and adults. Treatment of hydrocephalus usually involves surgical placement of a relocation system to drain the fluid into the abdominal cavity. Hydrocephalus may be caused by mechanical obstruction of the outflow of CSF from the ventricles or by faulty reabsorption. It can be also caused by CSF overproduction by the choroid plexus found in the lateral, third, and fourth ventricles of the brain. The choroid plexus is composed of a high resistance monolayer epithelium which surrounds a network of capillaries. Its primary function is to regulate transport of ions and water that control the production and movement of CSF. Therefore it is important to understand the mechanism of CSF production by the choroid plexus. Recently, a stable porcine choroid plexus (PCP-R) epithelial cell line with a high transepithelial resistance (TER) was developed that provides an important model to study regulation of CSF production. Ussing style electrophysiology was used to measure short circuit current (SCC) to characterize stimulated transepithelial ion transport in confluent PCP-R cells. GSK1016790, a TRPV4 agonist, was used to understand the role of TRPV4 in CSF production by the choroid plexus using PCP-R cell model. TRPV4 activation produces a sustained ion transport response that is consistent with an increase in cation secretion and/or anion absorption which is accompanied by a reversible decrease in TER. The effect of the agonist on both SCC and TER was blocked by HC067047, a TRPV4 antagonist, showing that the sustained ion transport and TER change is TRPV4 specific. TRPV4 mediated ion flux was inhibited by CFTR inhibitor II GlyH-101, a cell permeable inhibitor of the cAMP activated chloride channel CFTR, when added on either side of the membrane and was not accompanied by a TER reversal which showed that CFTR is activated by TRPV4 mediated ion flux. TMEM16A, a calcium activated chloride channel, was speculated to be located in that basal membrane as T16Ainh-AO1, a membrane permeable TMEM16A inhibitor, reversed the TRPV4 mediated ion flux when added on either side of the membrane. Slight reversal in TER was observed when T16Ainh-AO1 was added on the apical side. Apamin, a differential inhibitor of calcium activated small conductance potassium channel 1, 2 and 3 (SK1, SK2 and SK3) had no effect on the TRPV4 mediated ion flux. Whereas, fluoxetine, a membrane permeable inhibitor of SK1, SK2 and SK3 channel, inhibited the TRPV4 mediated ion flux and TER change. Bumetanide, an inhibitor of the sodium-potassium-chloride cotransporter reversed TRPV4 mediated ion flux when added on the apical membrane but not on the basal membrane indicating a possible K+ secretion via SK1 and/or SK4/IK channels and Cl- absorption through CFTR and TMEM16A channels. Acetazolamide, a carbonic anhydrase inhibitor and a compound used to treat hydrocephalus had no effect on the TRPV4 mediated ion flux. cAMP is an intracellular mediator involved in neuromodulator effects, inflammatory responses and other regulatory mechanisms and is constitutively activated by forskolin. In PCP-R cells, forskolin stimulated an increase in transepithelial ion flux that is consistent with an increase in cation absorption and/or anion secretion. Forskolin mediated ion transport was inhibited by CFTR inhibitor II GlyH-101 when added on either side of the membrane. No change in TER was observed. No effect on forskolin mediated ion flux was observed when T16Ainh-A01, apamin or fluoxetine were added. Forskolin stimulated transport is partially inhibited by 1 mM BaCl2. Barium chloride is a general inhibitor of K+ channels. No change in TER was observed.

Indiana University-Purdue University Indianapolis (IUPUI) / Pediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the Tmem67P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-andglucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using in vitro models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease.

La circulation pulmonaire est un système à faible pression (entre 10 et 15 mmHg au repos). Son principal rôle est la ré-oxygénation du sang qui permet l’apport aux organes du dioxygène nécessaire à leur fonctionnement. L’hyperTension Pulmonaire (HTP) est l’une de ses principales pathologies. Il s’agit d’une maladie rare engageant le pronostic vital du patient, définie par une pression artérielle pulmonaire moyenne supérieure ou égale à 25 mmHg au repos. Elle s’explique par une augmentation des résistances vasculaires pulmonaires liée, entre autres, au remodelage artériel présent dans cette pathologie participant à la diminution de la lumière artérielle. En effet, chez les patients atteint d’HTP, les trois tuniques formant l’artère (intima-media-adventice) voient leur structure se modifier. La plupart des travaux portent sur le remodelage de la media, mais de plus en plus d’études montrent que les premières altérations observées se situent au niveau de l’adventice. Plus précisément, les fibroblastes, les cellules majoritaires de cette tunique, agiraient comme régulateur clé de la fonction vasculaire pulmonaire. En réponse à un stress extérieur tel que l’hypoxie (à l’origine de certaines formes d'HTP), elles seraient les premières cellules à s’activer d'où leur dénomination par certains de « cellules sentinelles ». Cette activation se manifeste entre autres par des changements phénotypiques (différenciation en myofibroblastes), leur prolifération, leur migration, et la surproduction de protéines de la matrice extracellulaire. Ainsi les fibroblastes participent directement au remodelage artériel global observé dans l’HTP. Le calcium est connu pour réguler un grand nombre de voies de signalisation cellulaire impliquées dans les phénomènes précédemment cités. Au laboratoire, l’implication du canal TRPV4 (Transient Receptor Potential Vanilloid 4), un canal non sélectif perméable aux ions Ca2+, a déjà été montré concernant le remodelage de la media. L’activation du canal, amplifiée en situations pathologiques, participe à la migration et à la prolifération des cellules musculaires lisses des artères pulmonaires. Aussi, des données de la littérature montrent que TRPV4 joue un rôle important dans l’activité délétère des fibroblastes dans les sclérodermies, les fibroses pulmonaire et cardiaque. Il nous a donc semblé intéressant d’étudier l’implication des fibroblastes et du canal TRPV4 dans le remodelage adventitiel artériel lors de l’hypertension pulmonaire. Pour ce faire, nous nous sommes intéressés, dans une première partie, à l’implication du canal TRPV4 dans ce remodelage au niveau tissulaire. Nous avons ainsi montré, à l’aide de deux modèles animaux hypertendus, un induit par l’injection de monocrotaline et l’autre induit par une exposition hypoxique chronique, que la protéine TRPV4 est surexprimée dans l’adventice alors que son remodelage est significativement atténué chez des souris invalidées pour le gène trpv4. Dans un second temps, nous avons alors étudié le rôle de ce canal dans les réponses cellulaires impliquées dans le remodelage adventitiel. Pour cela nous avons mis au point la culture de fibroblastes d'adventice d'artères intrapulmonaires de rats. Puis par une approche pharmacologique (activateurs et bloqueurs du canal) et à l’aide de siRNA, nous avons montré que l’activation du canal TRPV4 favorise la prolifération (par incorporation de BrdU) et la migration (par test de brèche) des fibroblastes, ainsi que leur activité profibrotique avec la surproduction de matrice extracellulaire (MEC) (par quantification de l’expression protéique par Western blot). Il serait désormais intéressant de cultiver les fibroblastes en mimant in vitro les conditions pathologiques, en les plaçant dans un environnement hypoxique et/ou en les soumettant à un étirement chronique, et d’évaluer l’impact de l’HTP sur le canal et ses actions cellulaires. / Pulmonary circulation is a low pressure system (between 10 and 15 mmHg at rest). Its first role is blood oxygenation which allows to carry dioxygen to the organs fontionnality. Pulmonary Hypertension (PH) is one of the main pulmonary diseases. It is a rare and potentially fatal disorder, defined by a high arterial pulmonary mean pressure (greater than or equal to 25 mmHg at rest). This high pressure can be explained by the elevation of pulmonary arterial resistance and related to narrowing of the lumen of the artery, induced, among other, by the arterial remodeling in this pathology. Indeed, during the pathology implementation, the structure of the all three layers constituting the artery wall (intima-media-adventitia) is altered. The media and intima have received much attention from vascular biologists, howewer an increasing volume of experimental data indicates that this third compartment undergoes earlier and dramatic remodeling during PH. More specifically, the fibroblasts, the most abundant cells in adventitia, may act as key regulator of pulmonary vascular wall structure and function from the "outside-in". The fibroblasts may play the role of “sentinel cell” in the vessel wall. In responding to various stimuli, these cells are the first artery wall cells to show evidence of “activation” as proliferation, myofibroblast differenciation, migrationand invasion in the other wall layer, and extracellular matrix production. That way, fibroblasts participate directly to the overall artery remodeling observed in PH. Calcium is involved in numerous cellular signalling pathways such as those previously described. In the laboratory, we already proved that TRPV4 (Transient Receptor Potential Vanilloid) channel, a non-selective cationic channel calcium permeable, is involved in media remodeling. Moreover, several datas show that this channel play an important role in diseases in which we observe a negative role of fibroblast such as sclerodermia, cardiac and pulmonary fibrosis. Considering these results, we were interested in the role of TRPV4 in fibroblast during PH more precisely in the adventitial remodeling process observed in this pathology. We first demonstrated the involment of TRPV4 in the adventitia remodeling regarding the tissue. Using two different animal models of PH, chronic hypoxia and monocrotalin models, we identified that this protein was up-regulated in sick rats and the mouse knock-down for this gene developed attenuated PH and adventitia remodeling compare to the control. Then we studied the role of TRPV4 in the mechanism leading to the adventitia remodeling. Thanks to pharmacological molecule and siRNA we proved that activation of TRPV4 increased proliferation (BrdU assay), migration (wound assay) and fibrotic activity such as excessed production of extracellular matrix (using western blot analyse) of the fibroblasts. With all these results, it would be interested to culture fibroblasts in hypoxic conditions and/or subjecting themselves to chronicle stretch to imitate HTP pathology and evaluate TRPV4 role in these conditions