COMP promotes pancreatic fibrosis by activating pancreatic stellate cells through CD36-ERK/AKT signaling pathways.

BACKGROUND: Pancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP) and pancreatic stellate cells (PSCs) are the key cells of fibrosis. As an extracellular matrix (ECM) glycoprotein, cartilage oligomeric matrix protein (COMP) is critical for collagen assembly and ECM stability and recent studies showed that COMP exert promoting fibrosis effect in the skin, lungs and liver. However, the role of COMP in activation of PSCs and pancreatic fibrosis remain unclear. We aimed to investigate the role and specific mechanisms of COMP in regulating the profibrotic phenotype of PSCs and pancreatic fibrosis. METHODS: ELISA method was used to determine serum COMP in patients with CP. Mice model of CP was established by repeated intraperitoneal injection of cerulein and pancreatic fibrosis was evaluated by Hematoxylin-Eosin staining (H&E) and Sirius red staining. Immunohistochemical staining was used to detect the expression changes of COMP and fibrosis marker such as α-SMA and Fibronectin in pancreatic tissue of mice. Cell Counting Kit-8, Wound Healing and Transwell assessed the proliferation and migration of human pancreatic stellate cells (HPSCs). Western blotting, qRT-PCR and immunofluorescence staining were performed to detect the expression of fibrosis marker, AKT and MAPK family proteins in HPSCs. RNA-seq omics analysis as well as small interfering RNA of COMP, recombinant human COMP (rCOMP), MEK inhibitors and PI3K inhibitors were used to study the effect and mechanism of COMP on activation of HPSCs. RESULTS: ELISA showed that the expression of COMP significantly increased in the serum of CP patients. H&E and Sirius red staining analysis showed that there was a large amount of collagen deposition in the mice in the CP model group and high expression of COMP, α-SMA, Fibronectin and Vimentin were observed in fibrotic tissues. TGF-ß1 stimulates the activation of HPSCs and increases the expression of COMP. Knockdown of COMP inhibited proliferation and migration of HPSCs. Further, RNA-seq omics analysis and validation experiments in vitro showed that rCOMP could significantly promote the proliferation and activation of HPSCs, which may be due to promoting the phosphorylation of ERK and AKT through membrane protein receptor CD36. rCOMP simultaneously increased the expression of α-SMA, Fibronectin and Collagen I in HPSCs. CONCLUSION: In conclusion, this study showed that COMP was up-regulated in CP fibrotic tissues and COMP induced the activation, proliferation and migration of PSCs through the CD36-ERK/AKT signaling pathway. COMP may be a potential therapeutic candidate for the treatment of CP. Interfering with the expression of COMP or the communication between COMP and CD36 on PSCs may be the next direction for therapeutic research.

Biodistribution and therapeutic efficacy of a gold nanoparticle-based targeted drug delivery system against pancreatic cancer.

Pancreatic cancer is characterized by desmoplasia; crosstalk between pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) leads to the deposition of extracellular matrix proteins in the tumor environment resulting in poor vascularity. Targeting either PCCs or PSCs individually has produced mixed results, and there is currently no effective strategy to target both cell types simultaneously. Previously, we demonstrated, through in vitro cell culture experiments, that a specific gold nanoparticle-based nanoformulation containing the anti-EGFR antibody cetuximab (C225) as a targeting agent and gemcitabine as a chemotherapeutic agent effectively targets both PCCs and PSCs simultaneously. Herein, we extend our studies to test the ability of these in vitro tested nano formulations to inhibit tumor growth in an orthotopic co-implantation model of pancreatic cancer in vivo. Orthotopic tumors were established by co-implantation of equal numbers of PCCs and PSCs in the mouse pancreas. Among the various formulations tested, 5 nm gold nanoparticles coated with gemcitabine, cetuximab and poly-ethylene glycol (PEG) of molecular weight 1000 Da, which we named ACGP441000, demonstrated optimal efficacy in inhibiting tumor growth. The current study reveals an opportunity to target PCCs and PSCs simultaneously, by exploiting their overexpression of EGFR as a target, in order to inhibit pancreatic cancer growth.

Disruption of Super-Enhancers in Activated Pancreatic Stellate Cells Facilitates Chemotherapy and Immunotherapy in Pancreatic Cancer.

One major obstacle in the drug treatment of pancreatic ductal adenocarcinoma (PDAC) is its highly fibrotic tumor microenvironment, which is replete with activated pancreatic stellate cells (a-PSCs). These a-PSCs generate abundant extracellular matrix and secrete various cytokines to form biophysical and biochemical barriers, impeding drug access to tumor tissues. Therefore, it is imperative to develop a strategy for reversing PSC activation and thereby removing the barriers to facilitate PDAC drug treatment. Herein, by integrating chromatin immunoprecipitation (ChIP)-seq, Assays for Transposase-Accessible Chromatin (ATAC)-seq, and RNA-seq techniques, this work reveals that super-enhancers (SEs) promote the expression of various genes involved in PSC activation. Disruption of SE-associated transcription with JQ1 reverses the activated phenotype of a-PSCs and decreases stromal fibrosis in both orthotopic and patient-derived xenograft (PDX) models. More importantly, disruption of SEs by JQ1 treatments promotes vascularization, facilitates drug delivery, and alters the immune landscape in PDAC, thereby improving the efficacies of both chemotherapy (with gemcitabine) and immunotherapy (with IL-12). In summary, this study not only elucidates the contribution of SEs of a-PSCs in shaping the PDAC tumor microenvironment but also highlights that targeting SEs in a-PSCs may become a gate-opening strategy that benefits PDAC drug therapy by removing stromal barriers.

Pirfenidone alleviates chronic pancreatitis via suppressing the activation of pancreatic stellate cells and the M1 polarization of macrophages.

In the realm of fibroinflammatory conditions, chronic pancreatitis (CP) stands out as a particularly challenging ailment, lacking a dedicated, approved treatment. The potential of Pirfenidone (PFD), a drug originally used for treating idiopathic pulmonary fibrosis (IPF), in addressing CP's fibrotic aspects has sparked new interest. This investigation focused on the role of PFD in diminishing fibrosis and immune response in CP, using a mouse model induced by caerulein. The research extended to in vitro studies examining the influence of PFD on pancreatic stellate cells' (PSCs) behavior and the polarization of macrophages into M1 and M2 types. Advanced techniques like RNA sequencing and comprehensive data analyses were employed to decode the molecular interactions of PFD with PSCs. Supplementary experiments using techniques such as quantitative real-time PCR, western blotting, and immunofluorescence were also implemented. Results showed a notable reduction in pancreatic damage in PFD-treated mice, manifested through decreased acinar cell atrophy, lower collagen deposition, and a reduction in macrophage presence. Further investigation revealed PFD's capacity to hinder PSCs' migration, growth, and activation, alongside a reduction in the production and secretion of extracellular matrix proteins. This effect is primarily achieved by interfering with signaling pathways such as TGF-ß/Smad, Wnt/ß-catenin, and JAK/STAT. Additionally, PFD selectively hampers M1 macrophage polarization through the STAT3 pathway, without impacting M2 polarization. These outcomes highlight PFD's dual mechanism in moderating PSC activity and M1 macrophage polarization, positioning it as a promising candidate for CP therapy.

Retinoic acid signaling pathway in pancreatic stellate cells: Insight into the anti-fibrotic effect and mechanism.

Pancreatic stellate cells (PSCs) are activated following loss of cytoplasmic vitamin A (retinol)-containing lipid droplets, which is a key event in the process of fibrogenesis of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDCA). PSCs are the major source of cancer-associated fibroblasts (CAFs) that produce stroma to induce PDAC cancer cell growth, invasion, and metastasis. As an active metabolite of retinol, retinoic acid (RA) can regulate target gene expression in PSCs through its nuclear receptor complex (RAR/RXR or RXR/RXR) or transcriptional intermediary factor. Additionally, RA also has extranuclear and non-transcriptional effects. In vitro studies have shown that RA induces PSC deactivation which reduces extracellular matrix production through multiple modes of action, such as inhibiting TßRⅡ, PDGFRß, ß-catenin and Wnt production, downregulating ERK1/2 and JNK phosphorylation and suppressing active TGF-ß1 release. RA alone or in combination with other reagents have been demonstrated to have an effective anti-fibrotic effect on cerulein-induced mouse CP models in vivo studies. Clinical trial data have shown that repurposing all-trans retinoic acid (ATRA) as a stromal-targeting agent for human pancreatic cancer is safe and tolerable, suggesting the possibility of using RA for the treatment of CP and PDCA in humans. This review focuses on RA signaling pathways in PSCs and the effects and mechanisms of RA in PSC-mediated fibrogenesis as well as the anti-fibrotic and anti-tumor effects of RA targeting PSCs or CAFs in vitro and in vivo, highlighting the potential therapies of RA against CP and PDAC.

Meflin is a marker of pancreatic stellate cells involved in fibrosis and epithelial regeneration in the pancreas.

Pancreatic stellate cells (PSCs) are stromal cells in the pancreas that play an important role in pancreatic pathology. In chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC), PSCs are known to get activated to form myofibroblasts or cancer-associated fibroblasts (CAFs) that promote stromal fibroinflammatory reactions. However, previous studies on PSCs were mainly based on the findings obtained using ex vivo expanded PSCs, with few studies that addressed the significance of in situ tissue-resident PSCs using animal models. Their contributions to fibrotic reactions in CP and PDAC are also lesser-known. These limitations in our understanding of PSC biology have been attributed to the lack of specific molecular markers of PSCs. Herein, we established Meflin (Islr), a glycosylphosphatidylinositol-anchored membrane protein, as a PSC-specific marker in both mouse and human by using human pancreatic tissue samples and Meflin reporter mice. Meflin-positive (Meflin+ ) cells contain lipid droplets and express the conventional PSC marker Desmin in normal mouse pancreas, with some cells also positive for Gli1, the marker of pancreatic tissue-resident fibroblasts. Three-dimensional analysis of the cleared pancreas of Meflin reporter mice showed that Meflin+ PSCs have long and thin cytoplasmic protrusions, and are localised on the abluminal side of vessels in the normal pancreas. Lineage tracing experiments revealed that Meflin+ PSCs constitute one of the origins of fibroblasts and CAFs in CP and PDAC, respectively. In these diseases, Meflin+ PSC-derived fibroblasts showed a distinctive morphology and distribution from Meflin+ PSCs in the normal pancreas. Furthermore, we showed that the genetic depletion of Meflin+ PSCs accelerated fibrosis and attenuated epithelial regeneration and stromal R-spondin 3 expression, thereby implying that Meflin+ PSCs and their lineage cells may support tissue recovery and Wnt/R-spondin signalling after pancreatic injury and PDAC development. Together, these data indicate that Meflin may be a marker specific to tissue-resident PSCs and useful for studying their biology in both health and disease. © 2023 The Pathological Society of Great Britain and Ireland.

Therapeutic Strategies for Pancreatic-Cancer-Related Type 2 Diabetes Centered around Natural Products.

Pancreatic ductal adenocarcinoma (PDAC), a highly malignant neoplasm, is classified as one of the most severe and devastating types of cancer. PDAC is a notable malignancy that exhibits a discouraging prognosis and a rising occurrence. The interplay between diabetes and pancreatic cancer exhibits a reciprocal causation. The identified metabolic disorder has been observed to possess noteworthy consequences on health outcomes, resulting in elevated rates of morbidity. The principal mechanisms involve the suppression of the immune system, the activation of pancreatic stellate cells (PSCs), and the onset of systemic metabolic disease caused by dysfunction of the islets. From this point forward, it is important to recognize that pancreatic-cancer-related diabetes (PCRD) has the ability to increase the likelihood of developing pancreatic cancer. This highlights the complex relationship that exists between these two physiological states. Therefore, we investigated into the complex domain of PSCs, elucidating their intricate signaling pathways and the profound influence of chemokines on their behavior and final outcome. In order to surmount the obstacle of drug resistance and eliminate PDAC, researchers have undertaken extensive efforts to explore and cultivate novel natural compounds of the next generation. Additional investigation is necessary in order to comprehensively comprehend the effect of PCRD-mediated apoptosis on the progression and onset of PDAC through the utilization of natural compounds. This study aims to examine the potential anticancer properties of natural compounds in individuals with diabetes who are undergoing chemotherapy, targeted therapy, or immunotherapy. It is anticipated that these compounds will exhibit increased potency and possess enhanced pharmacological benefits. According to our research findings, it is indicated that naturally derived chemical compounds hold potential in the development of PDAC therapies that are both safe and efficacious.

A journey to and with the stars: The pancreatic stellate cell story.

The George E Palade Prize is the highest honour awarded by the International Association of Pancreatology, that recognises an individual who has made outstanding contributions to the understanding of the pancreas and pancreatic diseases. The 2023 Palade Prize was awarded to Professor Minoti Apte, University of New South Wales Sydney on September 16, 2023 during the Joint Meeting of the International Association of Pancreatology and the Indian Pancreas Club, held in Delhi, India. This paper summarises her Palade lecture wherein she reflects on her journey as a medical graduate, an academic and a researcher, with a particular focus on her team's pioneering work on pancreatic stellate cell biology and the role of these cells in health and disease. While there has been much progress in this field with the efforts of researchers worldwide, there is much still to be learned; thus it is a topic with ample scope for innovative research with the potential to translate into better outcomes for patients with pancreatic disease.

Arachidonate 15-lipoxygenase-mediated production of Resolvin D5n-3 DPA abrogates pancreatic stellate cell-induced cancer cell invasion.

Activation of pancreatic stellate cells (PSCs) to cancer-associated fibroblasts (CAFs) is responsible for the extensive desmoplastic reaction observed in PDAC stroma: a key driver of pancreatic ductal adenocarcinoma (PDAC) chemoresistance leading to poor prognosis. Specialized pro-resolving mediators (SPMs) are prime modulators of inflammation and its resolution, traditionally thought to be produced by immune cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipid mediator profiling PSCs as well as primary human CAFs express enzymes and receptors to produce and respond to SPMs. Human PSC/CAF SPM secretion profile can be modulated by rendering these cells activated [transforming growth factor beta (TGF-ß)] or quiescent [all-trans retinoic acid (ATRA)]. ATRA-induced nuclear translocation of arachidonate-15-lipoxygenase (ALOX15) was linked to increased production of n-3 docosapentaenoic acid-derived Resolvin D5 (RvD5n-3 DPA), among other SPMs. Inhibition of RvD5n-3 DPA formation increases cancer cell invasion, whereas addback of this molecule reduced activated PSC-mediated cancer cell invasion. We also observed that circulating concentrations of RvD5n-3 DPA levels were decreased in peripheral blood of metastatic PDAC patients when compared with those measured in plasma of non-metastatic PDAC patients. Together, these findings indicate that RvD5n-3 DPA may regulate cancer-stroma cross-talk and invasion.

Differential Effects of Pancreatic Cancer-Derived Extracellular Vesicles Driving a Suppressive Environment.

Pancreatic ductal adenocarcinoma (PDAC) cells display extensive crosstalk with their surrounding environment to regulate tumor growth, immune evasion, and metastasis. Recent advances have attributed many of these interactions to intercellular communication mediated by small extracellular vesicles (sEVs), involving cancer-associated fibroblasts (CAF). To explore the impact of sEVs on monocyte lineage transition as well as the expression of checkpoint receptors and activation markers, peripheral blood monocytes from healthy subjects were exposed to PDAC-derived sEVs. Additionally, to analyze the role of sEV-associated HA in immune regulation and tissue-resident fibroblasts, monocytes and pancreatic stellate cells were cultured in the presence of PDAC sEVs with or depleted of HA. Exposure of monocytes to sEVs resulted in unique phenotypic changes in HLA-DR, PD-L1, CD86 and CD64 expression, and cytokine secretion that was HA-independent except for IL-1ß and MIP1ß. In contrast, monocyte suppression of autologous T cell proliferation was reduced following exposure to HA-low sEVs. In addition, exposure of stellate cells to sEVs upregulated the secretion of various cytokines, including MMP-9, while removal of HA from PDAC-derived sEVs attenuated the secretion of MMP-9, demonstrating the role of sEV-associated HA in regulating expression of this pro-tumorigenic cytokine from stellate cells. This observation lends credence to the findings from the TCGA database that PDAC patients with high levels of enzymes in the HA synthesis pathway had worse survival rates compared with patients having low expression of these enzymes. PDAC-derived sEVs have an immune modulatory role affecting the activation state of monocyte subtypes. However, sEV-associated HA does not affect monocyte phenotype but alters cytokine secretion and suppression of autologous T cell proliferation and induces secretion of pro-tumorigenic factors by pancreatic stellate cells (PSC), as has been seen following the conversion of PSCs to cancer-associated fibroblasts (CAFs). Interruption of the hexosamine biosynthetic pathway, activated in PDAC producing the key substrate (UDP-GlcNAc) for HA synthesis, thus, represents a potential clinical interception strategy for PDAC patients. Findings warrant further investigations of underlying mechanisms involving larger sample cohorts.

Pancreatic Acinar Cells-Derived Sphingosine-1-Phosphate Contributes to Fibrosis of Chronic Pancreatitis via Inducing Autophagy and Activation of Pancreatic Stellate Cells.

BACKGROUND & AIMS: Studies have demonstrated that activated pancreatic stellate cells (PSCs) play a crucial role in pancreatic fibrogenesis in chronic pancreatitis (CP); however, the precise mechanism for PSCs activation has not been fully elucidated. We analyzed the role of injured pancreatic acinar cells (iPACs) in the activation of PSCs of CP. METHODS: Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling was evaluated in experimental CP induced by cerulein injection or pancreatic duct ligation, as well as in PACs injured by cholecystokinin. The activation of PSCs and pancreatic fibrosis in CP samples was evaluated by immunohistochemical and immunofluorescence analyses. In vitro coculture assay of iPACs and PSCs was created to evaluate the effect of the SPHK1/S1P pathway and S1P receptor 2 (SIPR2) on autophagy and activation of PSCs. The pathogenesis of CP was assessed in SPHK1-/- mice or PACs-specific SPHK1-knockdown mice with recombinant adeno-associated virus serotypes 9-SPHK1-knockdown, as well as in mice treated with inhibitor of SPHK1 and S1P receptor 2 (S1PR2). RESULTS: SPHK1/S1P was remarkably increased in iPACs and acinar cells in pancreatic tissues of CP mice. Meanwhile, the pathogenesis, fibrosis, and PSCs activation of CP was significantly prevented in SPHK1-/- mice and recombinant adeno-associated virus serotypes 9-SPHK1-knockdown mice. Meanwhile, iPACs obviously activated PSCs, which was prevented by SPHK1 knockdown in iPACs. Moreover, iPACs-derived S1P specifically combined to S1PR2 of PSCs, by which modulated 5' adenosine monophosphate-activated protein kinase/mechanistic target of rapamycin pathway and consequently induced autophagy and activation of PSCs. Furthermore, hypoxia-inducible factor 1-α and -2α promoted SPHK1 transcription of PACs under hypoxia conditions, which is a distinct characteristic of the CP microenvironment. Coincidently, inhibition of SPHK1 and S1PR2 activity with inhibitor PF-543 and JTE-013 obviously impeded pancreatic fibrogenesis of CP mice. CONCLUSIONS: The activated SPHK1/S1P pathway in iPACs induces autophagy and activation of PSCs by regulating the S1PR2/5' adenosine monophosphate-activated protein kinase/mammalian target of rapamycin pathway, which promotes fibrogenesis of CP. The hypoxia microenvironment might contribute to the cross talk between PACs and PSCs in pathogenesis of CP.

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.

A nanodrug simultaneously inhibits pancreatic stellate cell activation and regulatory T cell infiltration to promote the immunotherapy of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense extracellular matrix flooded with immune suppressive cells, resulting in extremely poor clinical response to immunotherapy. It has been revealed that the activation of pancreatic stellate cells (PSCs) makes considerable contributions to the immunological "cold" tumor microenvironment (TME). Herein, we developed a polyamino acid-based nanodrug incorporating the PSC activation inhibitor calcipotriol and anti-CXCL12 siRNA. The nanodrug was easily prepared with a small particle size and is capable of penetrating pancreatic tumors to inactivate PSCs and downregulate CXCL12. The in vivo results of orthotopic pancreatic tumor treatment demonstrated that codelivery of calcipotriol and anti-CXCL12 siRNA remodeled the PDAC TME with reduced extracellular matrix and decreased immunosuppressive T cells. Eventually, the infiltration of cytotoxic T cells was increased, thereby acting with immune checkpoint blockade (ICB) therapy for immunologically "cold" pancreatic tumors. In the present study, we propose a promising paradigm to improve the immunotherapy outcome of PDAC using nanodrugs that synchronously inhibit PSC activation and regulatory T-cell infiltration. STATEMENT OF SIGNIFICANCE: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense extracellular matrix (ECM) that impedes the tumor infiltration of therapeutic agents and cytotoxic T lymphocytes, resulting in a poor clinical response to immunotherapy. In the present study, we proposed a promising approach for enhanced immunotherapy of pancreatic cancer. Specifically, a nanodrug incorporating calcipotriol and anti-CXCL12 siRNA was synthesized to synchronously inactivate matrix-producing pancreatic stellate cells and suppress the infiltration of regulatory T cells. The reduced ECM removed the pathological barrier, preventing nanodrug penetration and effector T-cell infiltration, leading to a conversion of the immunosuppressive "cold" microenvironment to a "hot" microenvironment, which eventually boosted the immunotherapy of anti-PD-1 antibodies in pancreatic cancer.

Vitamin D3 analogue calcipotriol inhibits the profibrotic effects of transforming growth factor- ß1 on pancreatic stellate cells.

OBJECTIVE: To evaluate the inhibitory effect of vitamin D3 analogue calcipotriol (Cal) on the fibrosis of pancreatic stellate cells (PSCs) induced by TGF-ß1 and the rationality of Cal use in alcoholic chronic pancreatitis (ACP). MATERIAL AND METHODS: Double-labeling immunofluorescence was used for the identification of VDR+PSCs in the pancreas of healthy controls (HC) and ACP patients. Van Gieson staining for examination of collagen fibers. RT-qPCR and Western Blot for determining the mRNAs and proteins of VDR, TGF-ß1 and COL1A1 in the pancreas of ACP or in vitro PSCs. ELISA or LC-MS/MS for detection of serum TGF-ß1 and COL1A1 or 25(OH)D3. The PSC line (RP-2 cell) was used for the determination of proteomic alterations in Cal plus TGF-ß1 versus TGF-ß1 and to examine the effect of VDR gene knockdown. RESULTS: Enhanced expression of VDR was detected in RP-2 cells stimulated with alcohol (ALC) plus Cal versus Cal alone and in PSCs in the pancreas of ACP versus HC. The increased VDR+PSCs were positively correlated with the levels of COL1A1 mRNAs or areas of collagen deposition in the pancreas of ACP. TGF-ß1 was overexpressed in the pancreas of ACP and ALC-treated RP-2 cells while 25(OH)D3 level in serum was significantly decreased in ACP versus HC. Through a VDR-dependent mechanism, Cal antagonized 16 profibrotic proteins in TGF-ß1-induced RP-2 cells that included 7 extracellular matrix components, 2 cytoskeletal proteins, 2 fibrosis-associated factors (RUNX1 and TRAF2), TIMP-1, CCN1, integrin α11, an adhesion scaffold protein (TGFB1i1) and an enzyme mediating TGF-ß1-induced fibrogenesis (ENPP1). CONCLUSION: This study suggests that Cal administration may be a potential antifibrotic strategy via inhibiting TGF-ß1-mediated PSC action during the development of ACP.

Nintedanib Alleviates Chronic Pancreatitis by Inhibiting the Activation of Pancreatic Stellate Cells via the JAK/STAT3 and ERK1/2 Pathways.

BACKGROUND: Nintedanib (Ninte) has been approved for the treatment of pulmonary fibrosis, and whether it can ameliorate chronic pancreatitis (CP) is unknown. AIMS: This study was conducted to investigate the effect and molecular mechanism of Ninte on pancreatic fibrosis and inflammation in vivo and in vitro. METHODS: The caerulein-induced CP model of murine was applied, and Ninte was orally administered. Pathological changes in pancreas were evaluated using hematoxylin & eosin, Sirius Red, Masson's trichrome, and anti-Ki-67 staining. For in vitro studies, the effects of Ninte on cell viability, apoptosis, and migration of pancreatic stellate cells (PSCs) were determined by CCK-8, flow cytometry, and wound healing assays, respectively. The potential molecular mechanisms of the effects of Ninte on PSCs were analyzed by RNA-Seq and verified at the gene expression and protein activity levels by qRT-PCR and Western Blot. RESULTS: Ninte significantly alleviated the weight loss in mice with caerulein-induced CP and simultaneously attenuated the pancreatic damage, as evidenced by reduced acinar atrophy, collagen deposition, infiltration of inflammatory cells, and inhibited cell proliferation/regeneration. Besides, Ninte markedly suppressed the transcription of fibrogenic and proinflammatory genes in pancreatic tissues. Further in vitro studies showed that Ninte significantly inhibited the transcription and protein expression of genes corresponding to fibrogenesis and proliferation in PSCs. The results of RNA-Seq analysis and subsequent verification assays indicated that Ninte inhibited the activation and proliferation of PSCs via the JAK/STAT3 and ERK1/2 pathways. CONCLUSIONS: These findings indicate that Ninte may be a potential anti-inflammatory and anti-fibrotic therapeutic agent for CP.

The context-dependent role of the Na+/Ca2+-exchanger (NCX) in pancreatic stellate cell migration.

Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca2+ signaling and cellular Na+ loading. The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and - 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.

Pancreatic stellate cell-derived exosomal tRF-19-PNR8YPJZ promotes proliferation and mobility of pancreatic cancer through AXIN2.

The pancreatic stellate cells (PSCs) play an important role in the development of pancreatic cancer (PC) through mechanisms that remain unclear. Exosomes secreted from PSCs act as mediators for communication in PC. This study aimed to explore the role of PSC-derived exosomal small RNAs derived from tRNAs (tDRs) in PC cells. Exosomes from PSCs were extracted and used to detect their effects on PC cell proliferation, migration and invasion. Exosomal tDRs profiling was performed to identify PSC-derived exosomal tDRs. ISH and qRT-PCR were used to examine the tRF-19-PNR8YPJZ levels and clinical value in clinical samples. The biological function of exosomal tRF-19-PNR8YPJZ was determined using the CCK-8, clone formation, wound healing and transwell assays, subcutaneous tumour formation and lung metastatic models. The relationship between the selected exosomal tRF-19-PNR8YPJZ and AXIN2 was determined by RNA sequencing, luciferase reporter assay. PSC-derived exosomes promoted the proliferation, migration, and invasion of PC cells. Novel and abundant tDRs are found to be differentially expressed in PANC-1 cells after treatment with PSC-derived exosomes, such as tRF-19-PNR8YPJZ. PC tissue samples showed markedly higher levels of tRF-19-PNR8YPJZ than normal controls. Patients with PC exhibiting high tRF-19-PNR8YPJZ expression had a highly lymph node invasion, metastasis, perineural invasion, advanced clinical stage and poor overall survival. Exosomal tRF-19-PNR8YPJZ from PSCs targeted AXIN2 in PC cells and decreased its expression, thus activating the Wnt pathway and promoting proliferation and metastasis. Exosomal tRF-19-PNR8YPJZ from PSCs promoted proliferation and metastasis in PC cells via AXIN2.

Differential location of growth factors and pancreatic stellate cell activation in chronic pancreatitis.

Pancreatic fibrosis is characterized by the activation of pancreatic stellate cells leading to the expression of smooth muscle actin (α-SMA). Normal pancreatic tissue has predominantly quiescent stellate cells in periductal and perivascular locations, which do not express α-SMA. We aimed at studying the immunohistochemistry (IHC) expression pattern of α-SMA, platelet-derived growth factor (PDGF-BB) and transforming growth factor (TGF-ß) in the resected specimen of chronic pancreatitis. Twenty biopsies from resected specimens of patients with chronic pancreatitis were included. The expression was measured in comparison to positive control biopsies (breast carcinoma for PDGF-BB and TGF-ß and appendicular tissue for α-SMA) and scored based on a semi-quantitative system based on staining intensity. The percentage of positive cells was used for objective scoring, which ranged from 0 to 15. The scoring was done separately for acini, ducts, stroma and islet cell. All patients had undergone surgery for refractory pain and the median duration of symptoms was 48 months. On IHC, α-SMA was not expressed in the acini, ducts or islets, but had high expression in the stromal regions (vs. acini, ducts and islet, p < 0.05), TGF-ß1 was also expressed maximally in islet cells; however, the distribution among all locations was statistically similar. α-SMA expression in the pancreatic stroma is an indicator of the concentration of activated stellate cells in the stroma, a site for genesis of fibrosis under the influence of growth factors in the local milieu.

Toll-like receptor 5 tunes hepatic and pancreatic stellate cells activation.

OBJECTIVE: Stellate cells are responsible for liver and pancreas fibrosis and strictly correlate with tumourigenesis. Although their activation is reversible, an exacerbated signalling triggers chronic fibrosis. Toll-like receptors (TLRs) modulate stellate cells transition. TLR5 transduces the signal deriving by the binding to bacterial flagellin from invading mobile bacteria. DESIGN: Human hepatic and pancreatic stellate cells were activated by the administration of transforming growth factor-beta (TGF-ß). TLR5 was transiently knocked down by short-interference RNA transfection. Reverse Transcription-quantitativePCR and western blot were performed to analyse the transcript and protein level of TLR5 and the transition players. Fluorescence microscopy was performed to identify these targets in spheroids and in the sections of murine fibrotic liver. RESULTS: TGF-ß-activated human hepatic and pancreatic stellate cells showed an increase of TLR5 expression. TLR5 knockdown blocked the activation of those stellate cells. Furthermore, TLR5 busted during murine liver fibrosis and co-localised with the inducible Collagen I. Flagellin suppressed TLR5, COL1A1 and ACTA2 expression after the administration of TGF-ß. Instead, the antagonist of TLR5 did not block the effect of TGF-ß. Wortmannin, a specific AKT inhibitor, induced TLR5 but not COL1A1 and ACTA2 transcript and protein level. CONCLUSION: TGF-ß-mediated activation of hepatic and pancreatic stellate cells requires the over-expression of TLR5. Instead, its autonomous signalling inhibits the activation of the stellate cells, thus prompting a signalling through different regulatory pathways.

Pancreatic Stellate Cells and the Targeted Therapeutic Strategies in Chronic Pancreatitis.

Chronic pancreatitis (CP) is a disease characterized by inflammatory recurrence that accompanies the development of pancreatic fibrosis. As the mystery of CP pathogenesis is gradually revealed, accumulating evidence suggests that the activation of pancreatic stellate cells (PSCs) and the appearance of a myofibroblast-like phenotype are the key gatekeepers in the development of CP. Targeting PSCs to prevent their activation and conversion to a myofibroblast-like phenotype, as well as increasing antioxidant capacity to counteract ongoing oxidative stress, are effective strategies for preventing or treating CP. Therefore, we reviewed the crosstalk between CP and pancreatic fibrosis, summarized the activation mechanisms of PSCs, and investigated potential CP therapeutic strategies targeting PSCs, including, but not limited to, anti-fibrosis therapy, antioxidant therapy, and gene therapy. Meanwhile, the above therapeutic strategies are selected in order to update the available phytopharmaceuticals as novel complementary or alternative approaches for the prevention and treatment of CP to clarify their potential mechanisms of action and their relevant molecular targets, aiming to provide the most comprehensive therapeutic treatment direction for CP and to bring new hope to CP patients.