Site-specific genetic and functional signatures of aortic endothelial cells at aneurysm predilection sites in healthy and AngII ApoE-/- mice.

Aortic aneurysm is characterized by a pathological dilation at specific predilection sites of the vessel and potentially results in life-threatening vascular rupture. Herein, we established a modified "Häutchen method" for the local isolation of endothelial cells (ECs) from mouse aorta to analyze their spatial heterogeneity and potential role in site-specific disease development. When we compared ECs from aneurysm predilection sites of healthy mice with adjacent control segments we found regulation of genes related to extracellular matrix remodeling, angiogenesis and inflammation, all pathways playing a critical role in aneurysm development. We also detected enhanced cortical stiffness of the endothelium at these sites. Gene expression of ECs from aneurysms of the AngII ApoE-/- model when compared to sham animals mimicked expression patterns from predilection sites of healthy animals. Thus, this work highlights a striking genetic and functional regional heterogeneity in aortic ECs of healthy mice, which defines the location of aortic aneurysm formation in disease.

Endothelial Glycocalyx and Cardiomyocyte Damage Is Prevented by Recombinant Syndecan-1 in Acute Myocardial Infarction.

The outer layer of endothelial cells (ECs), consisting of the endothelial glycocalyx (eGC) and the cortex (CTX), provides a protective barrier against vascular diseases. Structural and functional impairments of their mechanical properties are recognized as hallmarks of endothelial dysfunction and can lead to cardiovascular events, such as acute myocardial infarction (AMI). This study investigated the effects of AMI on endothelial nanomechanics and function and the use of exogenous recombinant syndecan-1 (rSyn-1), a major component of the eGC, as recovering agent. ECs were exposed in vitro to serum samples collected from patients with AMI. In addition, in situ ECs of ex vivo aorta preparations derived from a mouse model for AMI were employed. Effects were quantified by using atomic force microscopy-based nanoindentation measurements, fluorescence staining, and histologic examination of the mouse hearts. AMI serum samples damaged eGC/CTX and augmented monocyte adhesion to the endothelial surface. In particular, the anaphylatoxins C3a and C5a played an important role in these processes. The impairment of endothelial function could be prevented by rSyn-1 treatment. In the mouse model of myocardial infarction, pretreatment with rSyn-1 alleviated eGC/CTX deterioration and reduced cardiomyocyte damage in histologic analyses. However, echocardiographic measurements did not indicate a functional benefit. These results provide new insights into the underlying mechanisms of AMI-induced endothelial dysfunction and perspectives for future studies on the benefit of rSyn-1 in post-AMI treatment.

Rac1 regulates lipid droplets formation, nanomechanical, and nanostructural changes induced by TNF in vascular endothelium in the isolated murine aorta.

Endothelial inflammation is recognized as a critical condition in the development of cardiovascular diseases. TNF-induced inflammation of endothelial cells is linked to the formation of lipid droplets, augmented cortical stiffness, and nanostructural endothelial plasma membrane remodelling, but the insight into the mechanism linking these responses is missing. In the present work, we determined the formation of lipid droplets (LDs), nanomechanical, and nanostructural responses in the model of TNF-activated vascular inflammation in the isolated murine aorta using Raman spectroscopy, fluorescence imaging, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We analysed the possible role of Rac1, a major regulator of cytoskeletal organization, in TNF-induced vascular inflammation. We demonstrated that the formation of LDs, polymerization of F-actin, alterations in cortical stiffness, and nanostructural protuberances in endothelial plasma membrane were mediated by the Rac1. In particular, we revealed a significant role for Rac1 in the regulation of the formation of highly unsaturated LDs formed in response to TNF. Inhibition of Rac1 also downregulated the overexpression of ICAM-1 induced by TNF, supporting the role of Rac1 in vascular inflammation. Altogether, our results demonstrate that LDs formation, an integral component of vascular inflammation, is activated by Rac1 that also regulates nanomechanical and nanostructural alterations linked to vascular inflammation.

Rapid shear stress-dependent ENaC membrane insertion is mediated by the endothelial glycocalyx and the mineralocorticoid receptor.

The contribution of the shear stress-sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR-/- vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.

Mineralocorticoid receptor-antagonism prevents COVID-19-dependent glycocalyx damage.

Proinflammatory cytokines target vascular endothelial cells during COVID-19 infections. In particular, the endothelial glycocalyx (eGC), a proteoglycan-rich layer on top of endothelial cells, was identified as a vulnerable, vasoprotective structure during infections. Thus, eGC damage can be seen as a hallmark in the development of endothelial dysfunction and inflammatory processes. Using sera derived from patients suffering from COVID-19, we could demonstrate that the eGC became progressively worse in relation to disease severity (mild vs severe course) and in correlation to IL-6 levels. This could be prevented by administering low doses of spironolactone, a well-known and highly specific aldosterone receptor antagonist. Our results confirm that SARS-CoV-2 infections cause eGC damage and endothelial dysfunction and we outline the underlying mechanisms and suggest potential therapeutic options.

AFM-based nanoindentation indicates an impaired cortical stiffness in the AAV-PCSK9DY atherosclerosis mouse model.

Investigating atherosclerosis and endothelial dysfunction has mainly become established in genetically modified ApoE-/- or LDL-R-/- mice transgenic models. A new AAV-PCSK9DYDY mouse model with no genetic modification has now been reported as an alternative atherosclerosis model. Here, we aimed to employ this AAV-PCSK9DY mouse model to quantify the mechanical stiffness of the endothelial surface, an accepted hallmark for endothelial dysfunction and forerunner for atherosclerosis. Ten-week-old male C57BL/6 N mice were injected with AAV-PCSK9DY (0.5, 1 or 5 × 1011 VG) or saline as controls and fed with Western diet (1.25% cholesterol) for 3 months. Total cholesterol (TC) and triglycerides (TG) were measured after 6 and 12 weeks. Aortic sections were used for atomic force microscopy (AFM) measurements or histological analysis using Oil-Red-O staining. Mechanical properties of in situ endothelial cells derived from ex vivo aorta preparations were quantified using AFM-based nanoindentation. Compared to controls, an increase in plasma TC and TG and extent of atherosclerosis was demonstrated in all groups of mice in a viral load-dependent manner. Cortical stiffness of controls was 1.305 pN/nm and increased (10%) in response to viral load (≥ 0.5 × 1011 VG) and positively correlated with the aortic plaque content and plasma TC and TG. For the first time, we show changes in the mechanical properties of the endothelial surface and thus the development of endothelial dysfunction in the AAV-PCSK9DY mouse model. Our results demonstrate that this model is highly suitable and represents a good alternative to the commonly used transgenic mouse models for studying atherosclerosis and other vascular pathologies.

Effects of Chronic Kidney Disease on Nanomechanics of the Endothelial Glycocalyx Are Mediated by the Mineralocorticoid Receptor.

Endothelial mechanics control vascular reactivity and are regulated by the mineralocorticoid receptor (MR) and its downstream target, the epithelial Na+ channel (ENaC). Endothelial dysfunction is a hallmark of chronic kidney disease (CKD), but its mechanisms are poorly understood. We hypothesized that CKD disrupts endothelial mechanics in an MR/ENaC-dependent process. METHODS: Primary human endothelial cells were cultured with uremic serum derived from children with stage 3-5 (predialysis) CKD or adult hemodialysis (HD) patients or healthy controls. The height and stiffness of the endothelial glycocalyx (eGC) and cortex were monitored by atomic force microscopy (AFM) using an ultrasensitive mechanical nanosensor. RESULTS: In a stage-dependent manner, sera from children with CKD induced a significant increase in eGC and cortex stiffness and an incremental reduction of the eGC height. AFM measurements were significantly associated with individual pulse wave velocity and serum concentrations of gut-derived uremic toxins. Serum from HD patients increased MR expression and mechanical stiffness of the endothelial cortex, an effect reversed by MR and ENaC antagonists, decreased eNOS expression and NO bioavailability, and augmented monocyte adhesion. CONCLUSION: These data indicate progressive structural damage of the endothelial surface with diminishing kidney function and identify the MR as a mediator of CKD-induced endothelial dysfunction.

Nanomechanics of the Endothelial Glycocalyx: From Structure to Function.

The negatively charged, brush-like glycocalyx covers the surface layer of endothelial cells. This layer of membrane-bound, carbohydrate-rich molecules covers the luminal surface of the endothelium along the entire vascular tree, mostly comprising glycoproteins and proteoglycans. Together with the underlying actin-rich endothelial cortex, 50 to 150 nm beneath the plasma membrane, the endothelial glycocalyx (eGC) is recognized as a vasoprotective nanobarrier and responsive hub. Importantly, both the eGC and cortex are highly dynamic and can adapt their nanomechanical properties (ie, stiffness and height) to changes in the environment. The constant change between a soft and a stiff endothelial surface is imperative for proper functioning of the endothelium. This review defines the nanomechanical properties of the eGC and stresses the underlying mechanisms and factors leading to a disturbed structure-function relationship. Specifically, under inflammatory conditions, the eGC is damaged, resulting in enhanced vascular permeability, tissue edema, augmented leukocyte adhesion, platelet aggregation, and dysregulated vasodilation. An integrated knowledge of the relationship between the nanomechanical properties, structure, and function of the eGC might be key in understanding vascular function and dysfunction. In this context, the clinical aspects for preservation and restoration of proper eGC nanomechanics are discussed, considering the eGC as a potentially promising diagnostic marker and therapeutic target in the near future.

Induction of ICER is superseded by smICER, challenging the impact of ICER under chronic beta-adrenergic stimulation.

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to ß-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged ß-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.

It takes more than two to tango: mechanosignaling of the endothelial surface.

The endothelial surface is a highly flexible signaling hub which is able to sense the hemodynamic forces of the streaming blood. The subsequent mechanosignaling is basically mediated by specific structures, like the endothelial glycocalyx building the top surface layer of endothelial cells as well as mechanosensitive ion channels within the endothelial plasma membrane. The mechanical properties of the endothelial cell surface are characterized by the dynamics of cytoskeletal proteins and play a key role in the process of signal transmission from the outside (lumen of the blood vessel) to the interior of the cell. Thus, the cell mechanics directly interact with the function of mechanosensitive structures and ion channels. To precisely maintain the vascular tone, a coordinated functional interdependency between endothelial cells and vascular smooth muscle cells is necessary. This is given by the fact that mechanosensitive ion channels are expressed in both cell types and that signals are transmitted via autocrine/paracrine mechanisms from layer to layer. Thus, the outer layer of the endothelial cells can be seen as important functional mechanosensitive and reactive cellular compartment. This review aims to describe the known mechanosensitive structures of the vessel building a bridge between the important role of physiological mechanosignaling and the proper vascular function. Since mutations and dysfunction of mechanosensitive proteins are linked to vascular pathologies such as hypertension, they play a potent role in the field of channelopathies and mechanomedicine.

A two-phase response of endothelial cells to hydrostatic pressure.

The vascular endothelium is exposed to three types of mechanical forces: blood flow-mediated shear stress, vessel diameter-dependent wall tension and hydrostatic pressure. Despite considerable variations of blood pressure during normal and pathological physiology, little is known about the acute molecular and cellular effects of hydrostatic pressure on endothelial cells. Here, we used a combination of quantitative fluorescence microscopy, atomic force microscopy and molecular perturbations to characterize the specific response of endothelial cells to application of pressure. We identified a two-phase response of endothelial cells with an initial response to acute (1 h) application of pressure (100 mmHg) followed by a different response to chronic (24 h) application. While both regimes induce cortical stiffening, the acute response is linked to Ca2+-mediated myosin activation, whereas the chronic cell response is dominated by increased cortical actin density and a loss in endothelial barrier function. GsMTx-4 and amiloride inhibit the acute pressure response, which suggests that the ENaC Na+ channel is a key player in endothelial pressure sensing. The described two-phase pressure response may participate in the differential effects of transient changes in blood pressure and hypertension.

The function of TRP channels in neutrophil granulocytes.

Neutrophil granulocytes are exposed to widely varying microenvironmental conditions when pursuing their physiological or pathophysiological functions such as fighting invading bacteria or infiltrating cancer tissue. Examples for harsh environmental challenges include among others mechanical shear stress during the recruitment from the vasculature or the hypoxic and acidotic conditions within the tumor microenvironment. Chemokine gradients, reactive oxygen species, pressure, matrix elasticity, and temperature can be added to the list of potential challenges. Transient receptor potential (TRP) channels serve as cellular sensors since they respond to many of the abovementioned environmental stimuli. The present review investigates the role of TRP channels in neutrophil granulocytes and their role in regulating and adapting neutrophil function to microenvironmental cues. Following a brief description of neutrophil functions, we provide an overview of the electrophysiological characterization of neutrophilic ion channels. We then summarize the function of individual TRP channels in neutrophil granulocytes with a focus on TRPC6 and TRPM2 channels. We close the review by discussing the impact of the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) on neutrophil granulocytes. Since neutrophil infiltration into PDAC tissue contributes to disease progression, we propose neutrophilic TRP channel blockade as a potential therapeutic option.

Role of TRPC1 channels in pressure-mediated activation of murine pancreatic stellate cells.

The tumor environment contributes importantly to tumor cell behavior and cancer progression. Aside from biochemical constituents, physical factors of the environment also influence the tumor. Growing evidence suggests that mechanics [e.g., tumor (stroma) elasticity, tissue pressure] are critical players of cancer progression. Underlying mechanobiological mechanisms involve among others the regulation of focal adhesion molecules, cytoskeletal modifications, and mechanosensitive (MS) ion channels of cancer- and tumor-associated cells. After reviewing the current concepts of cancer mechanobiology, we will focus on the canonical transient receptor potential 1 (TRPC1) channel and its role in mechano-signaling in tumor-associated pancreatic stellate cells (PSCs). PSCs are key players of pancreatic fibrosis, especially in cases of pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by the formation of a dense fibrotic stroma (desmoplasia), primarily formed by activated PSCs. Desmoplasia contributes to high pancreatic tissue pressure, which in turn activates PSCs, thereby perpetuating matrix deposition. Here, we investigated the role of the putatively mechanosensitive TRPC1 channels in murine PSCs exposed to elevated ambient pressure. Pressurization leads to inhibition of mRNA expression of MS ion channels. Migration of PSCs representing a readout of their activation is enhanced in pressurized PSCs. Knockout of TRPC1 leads to an attenuated phenotype. While TRPC1-mediated calcium influx is increased in wild-type PSCs after pressure incubation, loss of TRPC1 abolishes this effect. Our findings provide mechanistic insight how pressure, an important factor of the PDAC environment, contributes to PSC activation. TRPC1-mediated activation could be a potential target to disrupt the positive feedback of PSC activation and PDAC progression.

A novel intronic promoter of the Crem gene induces small ICER (smICER) isoforms.

The transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) regulate gene transcription in response to elevated cAMP levels. The Crem isoform inducible cAMP early repressor (Icer) is transcribed by the internal promoter P2 as a critical regulator of multiple cellular processes. Here, we describe a novel inducible Crem isoform, small Icer (smIcer), regulated by a newly identified promoter (P6). ChIP revealed binding of CREB to P6 in human and mouse myocardium. P6 activity was induced by constitutively active CREB or stimulation of adenylyl cyclase. In mice, smIcer mRNA was ubiquitously expressed and transiently induced by ß-adrenoceptor stimulation e.g., in heart and lung. SmICER repressed both basal and cAMP-induced activities of P6 and P2 promoters. Stimulation of adenylyl cyclase induced P2 and P6 in cell type-specific manner. Alternative translational start sites resulted in three different smICER proteins, linked to increased apoptosis sensitivity. In conclusion, the Crem gene provides two distinct and mutually controlled mechanisms of a cAMP-dependent induction of transcriptional repressors. Our results suggest not only that smICER is a novel regulator of cAMP-mediated gene regulation, but also emphasize that biological effects that have been ascribed solely to ICER, should be revised with regard to smICER.

Piezo1-induced durotaxis of pancreatic stellate cells depends on TRPC1 and TRPV4 channels.

Pancreatic stellate cells (PSCs) are primarily responsible for producing the stiff tumor tissue in pancreatic ductal adenocarcinoma (PDAC). Thereby, PSCs generate a stiffness gradient between the healthy pancreas and the tumor. This gradient induces durotaxis, a form of directional cell migration driven by differential stiffness. The molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the role of the ion channels Piezo1, TRPC1, and TRPV4 in PSC durotaxis. We found that PSC migration towards a stiffer substrate is diminished by altering Piezo1 activity. Moreover, disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. Hence, PSC durotaxis is optimal with an intermediary level of mechanosensitive channel activity, which we simulated using a numerically discretized mathematical model. Our findings suggest that mechanosensitive ion channels, particularly Piezo1, detect the mechanical microenvironment to guide PSC migration.

Prolonged Door-to-Balloon Time Leads to Endothelial Glycocalyx Damage and Endothelial Dysfunction in Patients with ST-Elevation Myocardial Infarction.

Damage to the endothelial glycocalyx (eGC) has been reported during acute ischemic events like ST-elevation myocardial infarction (STEMI). In STEMI, a door-to-balloon time (D2B) of <60 min was shown to reduce mortality and nonfatal complications. Here, we hypothesize that eGC condition is associated with D2B duration and endothelial function during STEMI. One hundred and twenty-six individuals were analyzed in this study (STEMI patients vs. age-/sex-matched healthy volunteers). After stimulating endothelial cells with patient/control sera, the eGC's nanomechanical properties (i.e., height/stiffness) were analyzed using the atomic force microscopy-based nanoindentation technique. eGC components were determined via ELISA, and measurements of nitric oxide levels (NO) were based on chemiluminescence. eGC height/stiffness (both p < 0.001), as well as NO concentration (p < 0.001), were reduced during STEMI. Notably, the D2B had a strong impact on the endothelial condition: a D2B > 60 min led to significantly higher serum concentrations of eGC components (syndecan-1: p < 0.001/heparan sulfate: p < 0.001/hyaluronic acid: p < 0.0001). A D2B > 60 min led to the pronounced loss of eGC height/stiffness (both, p < 0.001) with reduced NO concentrations (p < 0.01), activated the complement system (p < 0.001), and prolonged the hospital stay (p < 0.01). An increased D2B led to severe eGC shedding, with endothelial dysfunction in a temporal context. eGC components and pro-inflammatory mediators correlated with a prolonged D2B, indicating a time-dependent immune reaction during STEMI, with a decreased NO concentration. Thus, D2B is a crucial factor for eGC damage during STEMI. Clinical evaluation of the eGC condition might serve as an important predictor for the endothelial function of STEMI patients in the future.

Acid-base homeostasis orchestrated by NHE1 defines the pancreatic stellate cell phenotype in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) progresses in an organ with a unique pH landscape, where the stroma acidifies after each meal. We hypothesized that disrupting this pH landscape during PDAC progression triggers pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) to induce PDAC fibrosis. We revealed that alkaline environmental pH was sufficient to induce PSC differentiation to a myofibroblastic phenotype. We then mechanistically dissected this finding, focusing on the involvement of the Na+/H+ exchanger NHE1. Perturbing cellular pH homeostasis by inhibiting NHE1 with cariporide partially altered the myofibroblastic PSC phenotype. To show the relevance of this finding in vivo, we targeted NHE1 in murine PDAC (KPfC). Indeed, tumor fibrosis decreased when mice received the NHE1-inhibitor cariporide in addition to gemcitabine treatment. Moreover, the tumor immune infiltrate shifted from granulocyte rich to more lymphocytic. Taken together, our study provides mechanistic evidence on how the pancreatic pH landscape shapes pancreatic cancer through tuning PSC differentiation.

Simultaneous quantification of selected glycosaminoglycans by butanolysis-based derivatization and LC-SRM/MS analysis for assessing glycocalyx disruption in vitro and in vivo.

Glycosaminoglycans (GAGs) constitute the main building blocks of the endothelial glycocalyx (GLX), and disruption of GLX initiates and promotes endothelial dysfunction. Here, we aimed to develop a novel, specific and accurate LC-SRM/MS-based method for glycosaminoglycans (GAGs) profiling. The method involved butanolysis derivatization to facilitate GAG-specific disaccharide generation and its subsequent retention in LC-reversed-phase mode followed by mass spectrometric detection performed in positive ion-selected reaction monitoring (SRM) mode. GAG contents were measured in media of endothelial cells (EA.hy926) subjected to various GAG-degrading enzymes, as well as in murine plasma and urine in apolipoprotein E/low-density lipoprotein receptor-deficient (ApoE/LDLR -/-) mice and age-matched wild-type C57BL/6 mice. Alternatively, GLX disruption was verified by atomic force microscopy (AFM)-based analysis of GLX thickness. The proposed assay to quantify GAG-specific disaccharides presented high sensitivity for each of the analytes (LLOQ: 0.05-0.1 µg/mL) as well as accuracy and precision (86.8-114.9% and 2.0-14.3%, respectively). In medium of EA.hy926 cells subjected to GAG-degrading enzymes various GAG-specific disaccharides indicating the degradation of keratan sulphate (KS), heparan sulphate (HS), chondroitin sulphate (CHS) or hyaluronan (HA) were detected as predicted based on the characteristics of individual enzyme activity. In turn, AFM-based assessment of GLX thickness was reduced to a similar extent by all single enzyme treatments, whereas the most prominent reduction of GLX thickness was detected following the enzyme mixture. Plasma measurements of GAGs revealed age- and hypercholesterolemia-dependent decrease in GAGs concentration. In summary, a novel LC-SRM/MS-based method for GAG profiling was proposed that may inform on GLX status in cell culture for both in vitro and in vivo conditions.

TRPC1 channels regulate the activation of pancreatic stellate cells through ERK1/2 and SMAD2 pathways and perpetuate their pressure-mediated activation.

Pancreatic stellate cell (PSC) activation is a major event occurring during pancreatic ductal adenocarcinoma (PDAC) development. Up to now mechanisms underlying their activation by mechanical cues such as the elevated tissue pressure in PDAC remain poorly understood. Here we investigate the role of one potential mechano-transducer, TRPC1 ion channel, in PSC activation. Using pre-activated human siTRPC1 and murine TRPC1-KO PSCs, we show that TRPC1 promotes αSMA (α-smooth muscle actin) expression, the main activation marker, in cooperation with the phosphorylated SMAD2, under normal and elevated pressure. Functional studies following TRPC1 silencing demonstrate the dual role of TRPC1 in the modulation of PSC proliferation and IL-6 secretion through the activation of ERK1/2 and SMAD2 pathways. Moreover, pressurization changes the mechanical behavior of PSCs by increasing their cellular stiffness and emitted traction forces in a TRPC1-dependent manner. In summary, these results point to a role of TRPC1 channels in sensing and transducing the characteristic mechanical properties of the PDAC microenvironment in PSCs.