Background/Objectives
Chronic pancreatitis (CP) is a pathological syndrome of the pancreas characterized by fibrosis and inflammation. At present, there is no definitive ... More
Background/Objectives
Chronic pancreatitis (CP) is a pathological syndrome of the pancreas characterized by fibrosis and inflammation. At present, there is no definitive cure for CP. Sodium–glucose cotransporter-2 (SGLT2) inhibitors, a class of antidiabetic drugs, have been reported to exert antifibrotic effects in several organs. We evaluated the antifibrotic effect of the SGLT2 inhibitor canagliflozin on the pancreas.
Methods
C57BL/6 mice carrying a Kras mutation were intraperitoneally injected with caerulein to induce CP. Pancreatic tissues were collected after 8 weeks of feeding with or without canagliflozin and subsequently evaluated. The antifibrotic effects were also assessed in human pancreatic stellate cells (HPSCs) and in pancreatic tissue from patients with CP.
Results
Canagliflozin preserved pancreatic acinar cells and insulin–positive cells, and reduced pancreatic fibrosis in vivo. In HPSCs, canagliflozin suppressed proliferation, migration, and the expression of fibrotic markers and connective tissue growth factor (CTGF), unlike other SGLT2 inhibitors. A cytokine array was used to investigate the antifibrotic mechanism. Canagliflozin significantly downregulated osteoprotegerin (OPG), leading to the suppression of fibrotic markers and CTGF. Furthermore, canagliflozin activated AMP-activated protein kinase (AMPK), which contributed to the downregulation of OPG. This effect was also observed in pancreatic tissue in vivo by Western blot analysis. In addition, pancreatic tissue from patients with CP contained significantly more OPG-positive cells than did control pancreatic tissue. These results indicate that canagliflozin suppressed pancreatic fibrosis by downregulating OPG.
Conclusion
Canagliflozin may be an effective therapeutic agent for CP, and our data suggest the AMPK–OPG–CTGF axis is a novel target in CP. Less
The cryopreservation of primary human brain vascular pericytes (HBVPs) has facilitated their commercial availability as an essential component of the blood–brain barrie... More
The cryopreservation of primary human brain vascular pericytes (HBVPs) has facilitated their commercial availability as an essential component of the blood–brain barrier. Among the commercial suppliers of HBVPs in suspension, only one provides information regarding the cryoprotectant and vehicle used, i.e., 5% dimethyl sulfoxide (Me2SO) in complete medium, with no cryopreservation procedure given. Published protocols for the cryopreservation of brain vascular pericytes derived from human-induced pluripotent stem cells use traditional 10% Me2SO. In this study, we characterized the cryobiological response of HBVPs using graded freezing, both in the absence of cryoprotectants, which identifies their susceptibility to the main types of cryoinjury, and in the presence of several different cryoprotectants with the aim of quantifying post-thaw outcome. We first validated our graded freezing technique using human cerebral microvascular endothelial cell/D3 clone (hCMEC/D3) by comparing the membrane integrity results in this study in the absence and presence of 5% Me2SO plus 6% hydroxyethyl starch (HES) in hCMEC/D3 complete medium, with those previously published by our group. We then assessed the cryobiological response of HBVPs to controlled slow cooling at 1 °C/min in the absence and presence of various cryoprotectants, namely 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol, all in HBVP complete medium. We found that all cryoprotectants tested yielded immediate post-thaw membrane integrity >93%. Cryopreservation with 10% Me2SO resulted in 95.3 ± 0.7% membrane integrity; using a lower concentration of Me2SO (5%) alone, or in the presence of 6% HES, resulted in membrane integrities of 93.3 ± 0.8% and 94.2 ± 0.8%, respectively. For applications where avoiding Me2SO is preferred, we showed that HBVPs can be cryopreserved in 10% glycerol (post-thaw membrane integrity of 95.7 ± 0.5%). The AlamarBlue reduction assay with 3 h incubation time indicated that HBVPs cryopreserved with either 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol exhibited post-thaw metabolic activities which were not statistically different from those of unfrozen controls. Our study presents various application-specific options for cryopreservation of primary HBVPs with validated post-thaw membrane integrity and metabolic activity.
Keywords:
Human brain vascular pericytes, Graded freezing, Membrane integrity, Metabolic activity, Dimethyl sulfoxide, Hydroxyethyl starch, Glycerol, Fluorescence microscopy, Cryopreservation Less
Preterm premature rupture of membranes (PPROM) is a major cause of preterm birth and neonatal morbidity, with no established treatment to restore membrane integrity. Mese... More
Preterm premature rupture of membranes (PPROM) is a major cause of preterm birth and neonatal morbidity, with no established treatment to restore membrane integrity. Mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties, harbor promising therapeutic potential for fetal membranes repair. This study aimed to evaluate the rapid barrier reinforcement effect of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) using in vitro and ex vivo models of PPROM. A wound healing assay was conducted to assess the effect of WJ-MSCs on human amniotic epithelial cells proliferation and migration. An ex vivo PPROM model was established using incised human fetal membranes to simulate membrane rupture. WJ-MSCs were directly applied to the lesion site at varying doses. Therapeutic efficacy was evaluated by a leak test and histological analysis. WJ-MSCs and their conditioned medium significantly enhanced epithelial wound closure in vitro, demonstrating that soluble paracrine factors secreted by WJ-MSCs have the potential to aid membrane injury restoration. In the ex vivo PPROM model, WJ-MSCs treatment reduced both leakage area and frequency of fetal membranes, with the most significant effect observed at a medium dose (5.0×105 cells). Histological evaluations revealed partial recovery of the fetal membranes, particularly the chorion layer. These findings suggest that WJ-MSCs contribute to rapid barrier reinforcement of injured fetal membranes. Taken together, WJ-MSCs enhance epithelial healing and support restoration of membrane integrity, highlighting their potential as a therapeutic approach for PPROM. Further, in vivo studies are required to confirm therapeutic efficacy and evaluate safety. Less
The invasive and metastatic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and stroma, which include extracellular matrix ... More
The invasive and metastatic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and stroma, which include extracellular matrix (ECM) and cellular components, including cancer-associated fibroblasts (CAFs). Soluble factors secreted by cancer and stromal cells contribute to stroma remodeling through the secretion of ECM proteins, providing a favorable environment for cancer cell dissemination. The peptide endothelin-1 (ET-1), through two G protein-coupled receptors (GPCR), endothelin receptor type A (ETAR) and B (ETBR), acts on both cancer and stromal cells, engaging the protein β-arrestin1 (β-arr1), to bolster SOC progression. However, its role in the regulation of the ECM proteins by ovarian fibroblasts is not understood. This study delves into the role of ET-1 as a regulator of type I collagen (Col1) and fibronectin (FN). Less
The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response... More
The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. Borrelia burgdorferi (Bb), the causative bacteria of Lyme disease, is associated with neurological complications including meningitis–indeed, Bb has been isolated from the CSF of patients. While it is accepted that B. burgdorferi can enter the central nervous system (CNS) of patients, it is unknown how the bacteria crosses this barrier and how the pathogenesis of the disease leads to the observed symptoms in patients. We hypothesize that during infection Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state with the CNS parenchyma. Primary human choroid plexus epithelial cells were grown in culture and infected with B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. These results were validated via RT-qPCR. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models. Less
Fibroblastic reticular cells (FRCs) are stromal cells in secondary lymphoid organs, the major sites for HIV-1 infection of CD4+ T cells. Although FRCs regulate T cell sur... More
Fibroblastic reticular cells (FRCs) are stromal cells in secondary lymphoid organs, the major sites for HIV-1 infection of CD4+ T cells. Although FRCs regulate T cell survival, proliferation, and migration, whether they play any role in HIV-1 spread has not been studied. Here, we show that FRCs enhance HIV-1 spread via trans-infection in which FRCs capture HIV-1 and facilitate infection of T cells that come into contact with FRCs. FRCs mediate trans-infection in both two- and three-dimensional culture systems and in a manner dependent on the virus producer cells. This producer cell dependence, which was also observed for virus spread in secondary lymphoid tissues ex vivo, is accounted for by CD44 incorporated into virus particles and hyaluronan bound to such CD44 molecules. This virus-associated hyaluronan interacts with CD44 expressed on FRCs, thereby promoting virus capture by FRCs. Overall, our results reveal a novel role for FRCs in promoting HIV-1 spread. Less
Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional m... More
Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. Less
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy an... More
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples. Less
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy an... More
The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples. Less
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