Piezo1-mediated stellate cell activation causes pressure-induced pancreatic fibrosis in mice

Pancreatic fibrosis is a complication of chronic pancreatitis and is a prominent feature of pancreatic cancer. Pancreatic fibrosis is commonly observed in patients with prolonged pancreatic duct obstruction, which elevates intrapancreatic pressure. We show here that increased pancreatic duct pressure causes fibrosis and describes the mechanism by which pressure increases deposition of extracellular matrix proteins and fibrosis. We found that pancreatic stellate cells (PSCs), the source of the extracellular matrix proteins in fibrosis, express the mechanically activated ion channel Piezo1. By increasing intracellular calcium, mechanical stress or the Piezo1 agonist Yoda1-activated PSCs manifest by loss of perinuclear fat droplets and increased TGF-β1, fibronectin, and type I collagen expression. These effects were blocked by the Piezo1 inhibitor GsMTx4 and absent in PSCs from mice with conditional genetic deletion of Piezo1 in stellate cells, as was pancreatic duct ligation–induced fibrosis. Although TRPV4 has been proposed to have direct mechanosensing properties, we discovered that PSCs from Trpv4-KO mice were protected against Yoda1-triggered activation. Moreover, mice devoid of TRPV4 were protected from pancreatic duct ligation–induced fibrosis. Thus, high pressure within the pancreas stimulates Piezo1 channel opening, and subsequent activation of TRPV4 leads to stellate cell activation and pressure-induced chronic pancreatitis and fibrosis.


Introduction
Injury to the pancreas most commonly causes acute pancreatitis, which is accompanied by acinar cell necrosis (1-3); however, recurrent or sustained injury leads to chronic pancreatitis, which may be complicated by exocrine and endocrine insufficiency (4)(5)(6)(7). Chronic pancreatitis is a progressive inflammatory disease of the pancreas that is characterized by fibrosis and loss of insulin-producing β cells (6,8). Quality of life in patients with chronic pancreatitis is reduced due to abdominal pain, pancreatic exocrine insufficiency, and diabetes mellitus (7). Once pancreatic fibrosis develops, recovery of pancreatic function is limited (7). Several studies reported that people with chronic pancreatitis have reduced life expectancy (9), which may be due in part to increased risk of pancreatic cancer -a leading cause of cancer death (9,10).
Several factors contribute to the development and progression of chronic pancreatitis. Although alcohol consumption is the leading cause of chronic pancreatitis, genetic mutations and pancreatic duct obstruction are other well-recognized contributors (5,7,8). Each of these causes results in pancreatic fibrosis, which can lead to scarring and narrowing of large and small pancreatic ducts, both of which lead to increased intrapancreatic pressure. Thus, it appears that elevated duct pressure can cause pancreatic fibrosis and that pancreatic fibrosis can further exacerbate pancreatic pressure.
We recently demonstrated that pancreatic acinar cells sense pressure through the mechanically activated ion channel Piezo1 (11,12). Piezo1 is a large membrane-spanning protein that resides in many pressure-sensitive cells (13). Membrane tension caused by shear stress, membrane stretching, or high pressure opens the Piezo1 channel, allowing the influx of cations, mainly Ca 2+ (14)(15)(16). Excessive Piezo1 activation produces abnormally high concentrations of intracellular calcium ([Ca 2+ ] i ), which have deleterious effects on cells (11,13).
Pancreatic fibrosis results from the deposition of extracellular matrix (ECM) proteins that are secreted by activated pancreatic stellate cells (PSCs; refs. [17][18][19][20]. However, the mechanism by which force initiates Pancreatic fibrosis is a complication of chronic pancreatitis and is a prominent feature of pancreatic cancer. Pancreatic fibrosis is commonly observed in patients with prolonged pancreatic duct obstruction, which elevates intrapancreatic pressure. We show here that increased pancreatic duct pressure causes fibrosis and describes the mechanism by which pressure increases deposition of extracellular matrix proteins and fibrosis. We found that pancreatic stellate cells (PSCs), the source of the extracellular matrix proteins in fibrosis, express the mechanically activated ion channel Piezo1. By increasing intracellular calcium, mechanical stress or the Piezo1 agonist Yoda1-activated PSCs manifest by loss of perinuclear fat droplets and increased TGF-β1, fibronectin, and type I collagen expression. These effects were blocked by the Piezo1 inhibitor GsMTx4 and absent in PSCs from mice with conditional genetic deletion of Piezo1 in stellate cells, as was pancreatic duct ligation-induced fibrosis. Although TRPV4 has been proposed to have direct mechanosensing properties, we discovered that PSCs from Trpv4-KO mice were protected against Yoda1-triggered activation. Moreover, mice devoid of TRPV4 were protected from pancreatic duct ligationinduced fibrosis. Thus, high pressure within the pancreas stimulates Piezo1 channel opening, and subsequent activation of TRPV4 leads to stellate cell activation and pressure-induced chronic pancreatitis and fibrosis.
JCI Insight 2022;7(8):e158288 https://doi.org/10.1172/jci.insight.158288 these effects is unknown. Based on the observations that duct obstruction leads to pancreatic fibrosis, we postulated that PSCs are also pressure sensitive. We report here that PSCs express the mechanically sensitive ion channel Piezo1, which is responsible for pressure-induced pancreatic fibrosis.

Results
Increased intrapancreatic pressure causes pancreatic fibrosis. We developed a clinically relevant obstructive pancreatitis model by ligating the pancreatic tail region ( Figure 1A) to evaluate the effects of intrapancreatic pressure on fibrosis (21). Five minutes after ligation, pancreatic pressure in the tail region increased from 8.5 mmHg (unligated pressure) to 22.7 mmHg (ligated pressure) ( Figure 1B). Eight days after pancreatic duct ligation (PDL), substantial pancreatic fibrosis was observed in the tail region of the pancreata. The nonligated head region of the pancreas was unaffected (Figure 1, D-G and J, and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.158288DS1). Similar results were observed 30 days after duct ligation (Figure 1, H-K). Accompanying the high deposition of collagen was loss of endocrine and exocrine tissue in the tail region ( Figure 1, J and K).
Piezo1 GFAP -KO mice were protected from pressure-induced pancreatic fibrosis. The demonstration that duct obstruction induced pancreatic fibrosis raised the possibility that PSCs were pressure sensitive. Mechanically activated ion channels are key pressure sensors in many tissues, and we previously demonstrated that the most highly expressed mechanoreceptor in the pancreas is Piezo1 (11). To determine possible mechanoreceptor expression, Piezo1 was identified in PSCs by immunostaining ( Figure 1C and Supplemental Figure 2, A and B). To evaluate the potential role of Piezo1 in PSCs and pancreatic fibrosis, we deleted Piezo1 in cells expressing the stellate cell gene glial fibrillary acidic protein (GFAP). Piezo1 GFAP -KO mice were protected from partial duct ligation-induced pancreatic fibrosis 8 days after ligation (Figure 1, D-G). In contrast, mice with selective deletion of Piezo1 in pancreatic acinar cells (Piezo1 aci -KO mice) were not protected against pressure-induced fibrosis (Figure 1, D-G). Similarly, 30 days after duct ligation, substantial collagen deposition with loss of pancreatic exocrine and endocrine tissue was observed in WT but not in Piezo1 GFAP -KO mice (Figure 1, H-K). These findings indicate that pressure induced by duct ligation induces pancreatic fibrosis by activating stellate cells independently of acinar cell-mediated pancreatitis.
Piezo1 causes sustained [Ca 2+ ] i elevation in PSCs. It is well known that PSCs produce the excessive ECM proteins in pancreatic fibrosis, so we hypothesized that increased intrapancreatic pressure stimulates increased PSC ECM production. To investigate this possibility, we first cultured mouse PSCs on a Matrigel-coated plate to maintain a quiescent phenotype. As evidence of quiescence, nearly 97% of PSCs retained Bodipy + perinuclear fat droplets (Supplemental Figure 2C). To determine possible mechanoreceptor expression, Piezo1 was identified in PSCs by immunostaining ( Figure 1C Figure 2E) (23). Removing external calcium abolished the Yoda1-induced calcium influx, and replacing calcium (2 mM) restored calcium entry, proving that the Piezo1-mediated increase in [Ca 2+ ] i in PSCs was dependent on external calcium (Figure 2, F and G). High-dose Yoda1 (25 μM) did not affect PSC viability and membrane integrity. All cells responded to the calcium ionophore, ionomycin, after Yoda1 (25 μM) treatment (Supplemental Figure 3). Like mouse PSCs, Piezo1 channels are expressed in human PSCs and are sensitive to GsMTx4 blockade following Yoda1 (25 μM) stimulation ( Figure  2, H-J). Mechanical forces such as shear stress and pressure are physiological activators of Piezo1 (13,16). We applied mechanical force by touching PSCs with a glass pipette. Pressure applied for 1 second produced only a transient rise in [Ca 2+ ] i (Supplemental Figure 4). To apply mechanical force for longer periods of time and to avoid cell accommodations to prolonged mechanical pushing, we used fluid shear stress. Prolonged high fluid shear stress is an approach similar to high-fluid pressure situations during obstructive pancreatitis (21,24,25). To determine the effects of shear stress on human PSCs, we applied fluid shear stress at 12 dyne/cm 2 for 1 minute, which produced a sustained increase in [Ca 2+ ] i similar to that of Yoda1 (25 μM). These changes were blocked by GsMTx4 ( Figure 2, K and L). In contrast to the effects of high shear force, low shear stress (4 dyne/cm 2 for 1 minute) or high shear stress of shorter duration (12 dyne/cm 2   Piezo1 triggers PSC activation. In healthy pancreas, quiescent PSCs are characterized by perinuclear, vitamin A-containing fat droplets and low-level expression of ECM proteins such as fibronectin and collagen (17,(26)(27)(28). Upon activation, PSCs lose perinuclear fat droplets and produce excess ECM proteins. To determine if Piezo1 has the ability to convert quiescent PSCs to an activated phenotype, we applied Yoda1 (25 μM) to human PSCs. Within 24 hours, nearly 80% of PSCs had lost their perinuclear fat droplets and became elongated with a significant increase in mean cell area and maximum diameter (mean Feret's diameter-max; Figure 3, A-E) (29,30). Fibronectin, collagen type I, and Piezo1 mRNA levels were also significantly elevated (Figure 3, G, H, J, and K). After 4 days of Yoda1 treatment, fibronectin and collagen type I immunostaining were abundant ( Figure 3, F and I). Like human PSCs, Yoda1 converted mouse PSCs to an activated phenotype with reduced perinuclear fat droplets, changes in cell shape, and increased fibronectin and collagen type I ( Figure 4). All the effects of Yoda1 on fibrosis were prevented in Piezo1-deleted PSCs (Figure 4), confirming their Piezo1 dependence.
High shear stress induces PSC activation and fibrogenic responses in vitro. Application of high shear stress (12 dyne/cm 2 ) for 1 minute produced sustained [Ca 2+ ] i elevation in PSCs. To determine if physical force mediates stellate cell activation through Piezo1, we studied the effect of high fluid shear stress with and without the Piezo1 blocker, GsMTx4. Shear stress (12 dyne/cm 2 for 10 minutes) applied to human PSCs significantly reduced the number of perinuclear fat droplets ( Figure 5, A and B). Repeated injury to the pancreas, associated with edema and increased pancreatic pressure, leads to chronic pancreatitis (4,5,7,31). Our observation that Yoda1 increased Piezo1 expression ( Figure 3K) raised the possibility that repeated exposure to mechanical force may induce a Piezo1-mediated fibrogenic response. To test this hypothesis, we applied shear stress (25 dyne/cm 2 for 10 minutes) twice at an interval of 24 hours and examined the stellate cell activation and fibrogenic responses in vitro. Importantly, repeated shear stress increased fibronectin and collagen type I in human PSCs ( Figure 5, C-F). Treatment with the Piezo1 blocker, GsMTx4, attenuated the shear stress-mediated pathological changes ( Figure 5), demonstrating that mechanical force induced stellate cell activation and increased ECM protein synthesis through Piezo1.
Piezo1 signaling mediates TRPV4 channel opening in PSCs. Recently, we discovered that Piezo1 downstream signaling activates the TRPV4 channel in pancreatic acinar cells and human umbilical vein endothelial cells (HUVECs) (13,24). Here we detected functional TRPV4 channels in mouse and human PSCs ( Figure 6, A-F). The TRPV4 agonist, GSK1016790A, produced a sustained elevation in [Ca 2+ ] i in quiescent mouse and human PSCs that was blocked with the TRPV4 blocker HC067047 ( Figure 6, B, C, E, and F) (13). Although, under certain circumstances, TRPV4-expressing cells respond to mechanical force, this appears to be an indirect effect, since it has not been demonstrated that mechanical manipulations directly cause TRPV4 channel opening (13,14,24,32). To determine if TRPV4 is involved in Piezo1-mediated changes in [Ca 2+ ] i in PSCs, we isolated cells from TRPV4-KO mice. Yoda1 (25 μM) or prolonged, high shear stress (12 dyne/cm 2 for 1 minute), which normally produce sustained elevations in [Ca 2+ ] i , caused only transient calcium elevations in TRPV4-null PSCs ( Figure 6, G-I, L, and M). 5′,6′-Epoxyeicosatrienoic acid is an endogenous activator of the TRPV4 channel produced from arachidonic acid via a PLA2-AA cytochrome P450 epoxygenase-dependent pathway (32,33). To determine if Piezo1 activates PLA2-AA, we used secretory and cytosolic PLA2 inhibitors YM26734 and AACOCF3 (13). Together, YM26734 and AACOCF3 significantly inhibited the Yoda1-mediated sustained elevation in [Ca 2+ ] i in PSCs from WT mice ( Figure 6, J and K). Additionally, GsMTx4, blocked the effects of high shear stress (12 dyne/cm 2 for 1 minute) on the transient [Ca 2+ ] i elevation in TRPV4-null PSCs, confirming that the initial transient calcium influx was due to Piezo1, while subsequent TRPV4 channel opening was responsible for the sustained rise in [Ca 2+ ] i ( Figure 6, N and O).
TRPV4-KO mice were protected from pressure-induced pancreatic fibrosis. Having determined that the sustained elevation in [Ca 2+ ] i produced by Piezo1 activation requires TRPV4 opening, we proposed that TRPV4 was responsible for stellate cell activation. To test this possibility, we treated PSCs isolated from TRPV4-KO mice with Yoda1 (25 μM). In contrast to WT PSCs, Yoda1 did not activate PSCs from TRPV4-KO mice, and no changes in PSC activation parameters (cell shape, perinuclear fat droplet abundance, and ECM protein expression) were observed (Figure 7, A-I). If pathological effects of Piezo1 (high-pressure-induced stellate cell activation and fibrosis) require TRPV4 channels, we would expect that of WT and Piezo1 GFAP -KO mice (n = 3-6). Statistical analyses were calculated using 2-tailed Student's t test for 2 groups, and multiple groups were analyzed by 1-way ANOVA. **P ≤ 0.01, ***P ≤ 0.001; ****P ≤ 0.0001. Scale bar: 100 μm.

Discussion
Pancreatic fibrosis is an irreversible complication of chronic pancreatitis that is often accompanied by loss of endocrine and exocrine function (6)(7)(8). Fibrosis is composed of ECM produced by activated PSCs, which also secrete proinflammatory cytokines that may amplify pancreatic inflammation and accelerate the loss of acinar and islet cells (34). Pancreatic fibrosis also increases the risk of pancreatic ductal adenocarcinoma (PDAC) (10). The dense desmoplasia found in PDAC is a product of a subtype of activated PSCs known as cancer-associated fibroblasts and poses a major hurdle for chemotherapeutic-based drug delivery. Effective antifibrotic therapies are lacking; therefore, most current efforts are directed at preventing fibrosis by blocking PSC activation. The 2 most common factors leading to chronic pancreatitis are heavy alcohol use and conditions producing sustained elevations in pancreatic duct pressure, such as duct strictures, cysts, pseudocysts, and obstructive tumors. We show here that PSCs exhibit pressure sensitivity by virtue of their expression of the mechanically activated ion channel Piezo1 and that activation of Piezo1 initiates a fibrogenic response. Complete manifestation of the pathological consequences of Piezo1 activation requires calcium-triggered TRPV4 channel opening and its accompanying calcium influx.
The current study demonstrates that increased intraductal pressure causes pancreatic fibrosis mediated by PSCs and that PSC sensitivity to pressure is mediated by Piezo1 activation. Brief high shear stress or low shear stress for longer times produced transient increases in [Ca 2+ ] i that were insufficient to activate PSCs. In contrast, Yoda1 or sustained higher shear force produced a sustained elevation in [Ca 2+ ] i and induced PSC activation, manifest by cell elongation, loss of perinuclear fat droplets, and stimulation of profibrotic TGF-β1 and ECM protein (e.g., fibronectin and collagen type I) gene expression. Our findings that Piezo1 GFAP -KO mice were protected from pressure-induced fibrosis suggest that a Piezo1 blocker could be a possible treatment for pancreatic fibrosis.
Pancreatic fibrosis is an active inflammatory process, accompanied by cell-to-cell contact and dynamic production of inflammatory molecules (35). Activated PSCs secrete IL-6, IL-1β, monocyte-specific chemokine (MCP-1), and TNF-α; activate tissue-resident macrophages; and recruit inflammatory monocytes, which are major regulators of fibrosis (35)(36)(37)(38)(39). In human fibrotic tissue and a rat model of chronic pancreatitis, macrophages are in close proximity to PSCs (36) and exacerbate the progression of pancreatic fibrosis through the production of TNF-α and TGF-β1 (36). Importantly, mechanical forces generate proinflammatory responses in macrophages and monocytes in a Piezo1-dependent manner (40)(41)(42)(43). This is illustrated by the finding that macrophages lacking Piezo1 exhibited decreased inflammation and enhanced wound healing (43). Piezo1 signaling in myeloid cells also exacerbated a mouse model of pulmonary fibrosis (41,44); thus, it appears that Piezo1 can induce fibrosis by acting both directly on stellate cells and indirectly through inflammatory cells. It is conceivable that a blocker of Piezo1 or its downstream signaling pathways could inhibit stellate cell activation and reduce the fibrogenic responses triggered by inflammatory immune cells.
We recently reported that pancreatic acinar cells express Piezo1 and that elevated pancreatic pressure can cause pancreatitis (11,24). During the course of pancreatitis, stellate cells can be activated by proinflammatory molecules, some of which are generated by acinar cells (27,39,(45)(46)(47). Thus, it is possible that fibrosis may result from pressure acting directly on PSCs or indirectly on acinar cells through the induction of pancreatitis and subsequent stellate cell activation. In the current study, we observed that PDL produced Data are presented as mean ± SEM. Statistical analyses were calculated using 2-tailed Student's t test for 2 groups, and multiple groups were analyzed by 1-way ANOVA. *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001; ****P ≤ 0.0001. Scale bar: 10 μm.  G, H, J, and K) mRNA levels of fibronectin, collagen type I, TGF-β1, and Piezo1 in human PSCs 24 hours after treatment with Yoda1 (25 μM) (3-5 experiments). Data are shown as mean ± SEM. Scale bars: 10 μm. Statistical analyses were calculated using 2-tailed Student's t test for 2 groups, and multiple groups were analyzed by 1-way ANOVA. *P ≤ 0.05; **P ≤ 0.01, ****P ≤ 0.0001. extensive pancreatic fibrosis in pancreata of WT and Piezo1 aci -KO mice, but not Piezo1 GFAP -KO mice, indicating that Piezo1 channels in stellate cells rather than acinar cells are responsible for pressure-induced fibrosis. Although acinar cell-mediated inflammatory signaling undoubtedly contributes to the development of chronic pancreatic and fibrosis under certain conditions (46,47), it appears that elevated pancreatic pressure directly promotes stellate cell activation and fibrosis.
TGF-β1 is a major profibrogenic cytokine and a target for antifibrotic therapies (48,49). Regulation of TGF-β1 function depends on site-specific activation by integrins (50)(51)(52), and recently, it has been demonstrated that mechanical activation of Piezo1 converts inactive integrins to an active form (50)(51)(52). In addition to the role of Piezo1 in activation of TGF-β1, our results demonstrate that Piezo1 increases TGF-β1 at the transcriptional level.
Discovery of TRPV4 in PSCs raised the possibility that the pathological effects of Piezo1 on generation of fibrosis may require TRPV4 (53, 54). Our results indicate that Piezo1 senses mechanical force and initiates calcium signaling, resulting in TRPV4 activation. In the absence of TRPV4, mechanical force or Yoda1 did not generate the sustained eleva tion in [Ca 2+ ] i that was necessary to alter PSC morphology, modify perinuclear fat droplet abundance, or initiate fibrogenic responses. Although under certain circumstances, TRPV4 has been variously reported as mechanosensitive, this has not been demonstrated in cell-free systems (14,55,56), and it seems likely that mechanoreceptor properties that were attributed to TRPV4 may be due to true mechanically activated ion channels like Piezo1 that happen to be coexpressed (11,13,24). PSCs appear to be another example of Piezo1 and TRPV4 interdependence.
The observation that Yoda1 elevated Piezo1 and TRPV4 mRNA levels in PSCs from WT mice suggests that prolonged pressure may increase the fibrogenic response ( Figure 3K and Supplemental Figure 5). It will be interesting to evaluate Piezo1 and TRPV4 in stellate cells of patients with chronic obstructive pancreatitis and to determine if administration of a Piezo1 or TRPV4 blocker can block or reverse obstructive chronic pancreatitis and fibrosis in humans. TRPV4 is expressed in many organs that have mechanosensing properties and exhibit fibrosis in response to injury including the heart, lung, kidney, liver, skin, and intestine (57-59). In the liver, TRPV4 expression was increased in hepatic fibrosis and linked to TGF-β1-induced hepatic stellate cell activation. Like hepatic fibrosis, TRPV4 was upregulated in fibrotic pulmonary tissue and TRPV4-KO mice were protected from pulmonary fibrosis (59). In the heart, TRPV4 converts fibroblasts to a myofibroblast phenotype through a TGF-β1-mediated pathway (57). Thus, TRPV4 is an established mediator of tissue fibrosis. In addition  to our findings in the pancreas, TRPV4 expression was found to be upregulated in a model of alcoholand high-fat diet-induced pancreatitis (53), and TRPV4 is expressed in macrophages and linked with inflammation (60)(61)(62). Our observations together with these findings support a possible strategy for preventing or treating pancreatic fibrosis by blocking Piezo1 or TRPV4 channels. In vivo experiments. PDL of the tail region was performed as previously described (21,24). The pancreas was visualized using a stereomicroscope, and the tail region of the main pancreatic duct was ligated with 7-0 (0.5 metric) nonabsorbable, Prolene suture without damaging underlying arteries and veins. Mice suffering injury to any underlying blood vessels were excluded from the experiment. Mice were sacrificed at day 8 or day 30 after surgery.

Animals
In vitro experiments. Mouse and human PSCs were isolated using collagenase digestion (26). Modified Krebs Henseleit Buffer (KHB) solution (100 mL) was prepared as described previously (11). Pancreatic tissue was digested with 2 mg of collagenase NB 8 (SERVA, catalog 17456) dissolved in 10 mL of modified KHB solution containing 1 mg soybean trypsin inhibitor (SBTI 1-S; MilliporeSigma, catalog T9003) and 20 mg BSA (Thermo Fisher Scientific; BP1600-100).Digestion solution (5 mL) was used to inflate the pancreas. Pancreas with 5 mL of digestion solution was incubated in a shaking water bath for 10 minutes at 37°C. The solution was then discarded. The pancreas tissue was cut into small pieces and digested with fresh 5 mL digestion buffer. Tissue was incubated in a shaking water bath for 40 minutes at 37°C. The cells were then separated from tissue by pipetting up-down with a 10 mL pipette and passed sequentially through a 70 μm and a 40 μm cell strainer. The filtrate was centrifuged at 150g for 3 minutes at room temperature. The cells were washed with 10 mL Leibovitz's media (Thermo Fisher Scientific, catalog 11415-064), passed through a 20 μm filter, and centrifuged at 80g for 3 minutes at room temperature. The supernate was removed, and isolated stellate cells were plated on a thin-layered Matrigel-coated glass bottom culture plate (MatTek, P35G-0-14-C). Before plating the cells, Matrigel solution (Corning, catalog 354234) at a ratio of 1.5:100 DMEM/F12 (Thermo Fisher Scientific, catalog 11330-032) was poured onto the culture plate and incubated for 2 hours at 37°C to form a thin layer of Matrigel coating. Matrigel mixture was removed, and the plate was washed with PBS before cells were plated. Cell culture media, DMEM/F12 with 5% FBS was used for mouse stellate cells. Fresh, human pancreatic tissue (provided by Duke University's BioRepository & Precision Pathology Center under IRB approval) was digested with collagenase as described above with modifications. Collagenase (2.5 mg) in 10 mL of modified KHB solution was used for digestion. The digested tissues were filtered through a 100 μm cell strainer. The cells were cultured with DMEM/F12 with 10% FBS in a Matrigel-coated plate. After 24 hours, the cell media was replaced with fresh media to remove unattached and dead cells. After 2 days, cells were immunostained for GFAP and used for experiments. Perinuclear fat droplets were stained with BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene; Invitrogen, catalog D3922) collagen type I and fibronectin staining for the data shown in F and G. (J-M) Pancreatic duct ligation (PDL) at the tail region of the pancreas induced chronic pancreatitis and fibrosis in WT and TRPV4-KO mice. Eight days after PDL, chronic pancreatitis and fibrosis parameters of the tail region included (J) H&E staining, (K) H&E score, (L) Masson's trichrome staining, and (M) area of WT and TRPV4-KO mice (n = 5). Statistical comparisons were made using 2-tailed Student's t test. ***P ≤ 0.001. Scale bar: 100 μm.