Expression Of HGF Research Articles

The Expression Quantitative Trait Loci in Immune Response Genes Impact the Characteristics and Survival of Colorectal Cancer.

The impact of germline variants on the regulation of the expression of tumor microenvironment (TME)-based immune response genes remains unclear. Expression quantitative trait loci (eQTL) provide insight into the effect of downstream target genes (eGenes) regulated by germline-associated variants (eVariants). Through eQTL analyses, we illustrated the relationships between germline eVariants, TME-based immune response eGenes, and clinical outcomes. In this study, both RNA sequencing data from primary tumor and germline whole-genome sequencing data were collected from patients with stage III colorectal cancer (CRC). Ninety-nine high-risk subjects were subjected to immune response gene expression analyses. Seventy-seven subjects remained for further analysis after quality control, of which twenty-two patients (28.5%) experienced tumor recurrence. We found that 65 eQTL, including 60 germline eVariants and 22 TME-based eGenes, impacted the survival of cancer patients. For the recurrence prediction model, 41 differentially expressed genes (DEGs) achieved the best area under the receiver operating characteristic curve of 0.93. In total, 19 survival-associated eGenes were identified among the DEGs. Most of these genes were related to the regulation of lymphocytes and cytokines. A high expression of HGF, CCR5, IL18, FCER1G, TDO2, IFITM2, and LAPTM5 was significantly associated with a poor prognosis. In addition, the FCER1G eGene was associated with tumor invasion, tumor nodal stage, and tumor site. The eVariants that regulate the TME-based expression of FCER1G, including rs2118867 and rs12124509, were determined to influence survival and chromatin binding preferences. We also demonstrated that FCER1G and co-expressed genes in TME were related to the aggregation of leukocytes via pathway analysis. By analyzing the eQTL from the cancer genome using germline variants and TME-based RNA sequencing, we identified the eQTL in immune response genes that impact colorectal cancer characteristics and survival.

CircPRKCH modulates extracellular matrix formation and metabolism by regulating the miR-145/HGF axis in osteoarthritis

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease. Extracellular matrix (ECM) degradation is essential for OA progression. Previous studies have shown that circular RNAs (circRNAs) are involved in the pathological process of OA. CircPRKCH has been shown to be upregulated in OA chondrocytes. The present study was aimed to explore the roles of circPRKCH in vivo and in vitro models of OA and its underlying molecular mechanisms.MethodsIL-1β-induced chondrocytes and mice injected with monosodium iodoacetate were used as OA models in vitro and in vivo, respectively. RT-qPCR was performed to measure the expression of circPRKCH, miR-145, and HGF in cartilage tissues and chondrocytes. The interaction between miR-145 and circPRKCH or HGF was verified by a dual-luciferase reporter assay. Chondrocyte apoptosis, viability, and ECM-related proteins were examined by flow cytometry, MTT assay, and Western blotting, respectively. Histopathological changes were detected by HE and Safranin O-fast green staining.ResultsThe expression of circPRKCH and HGF was increased in OA cartilage tissues and IL-1β-treated chondrocytes, while miR-145 expression was decreased. IL-1β induced chondrocyte apoptosis and ECM degradation in chondrocytes. Moreover, circPRKCH promoted HGF expression and activated HGF/c-MET by directly binding to miR-145. miR-145 knockdown or HGF overexpression significantly reversed circPRKCH knockdown-mediated inhibition of apoptosis and ECM degradation in IL-1β-induced chondrocytes. Besides, miR-145 overexpression alleviated IL-1β-induced chondrocyte apoptosis and ECM degradation by inhibiting HGF/c-MET. Finally, circPRKCH knockdown reduced ECM degradation by regulating the miR-145/HGF axis in an experimental OA model in mice.ConclusionOur study demonstrated that circPRKCH promoted chondrocyte apoptosis and ECM degradation via the miR-145/HGF axis in OA, which may provide a novel target for OA treatment.

The relationship between clinical parameters and hepatocyte growth factor/c-Met levels in the sperm of infertile males

Objective: Hepatocyte growth factor is a pleiotropic cytokine with potent mitogenic activity in many organs and tissues. The receptor for hepatocyte growth factor is c-Met, a transmembrane glycoprotein with tyrosine kinase activity. We investigated the relationships between hepatocyte growth factor and c-Met expression levels on sperm motility and male infertility. Methods: The study was designed as a prospective cohort, single-center clinical trial and was conducted between March 2015 and June 2016. The control group consisted of 31 healthy male volunteers with children, while the study group consisted of 61 men who were diagnosed with male infertility after presenting to the In Vitro Fertilization Unit of our Institute. All participants in the study were aged 18–60 years. Sperm samples were taken from each participant and divided in two. The fresh sample was examined immediately for expression of c-Met, while the other was stored frozen and evaluated later for hepatocyte growth factor. Primary outcome measures were the levels of hepatocyte growth factor and c-Met expression, while secondary outcome measures were the correlation of these levels with sperm count, morphology and motility. Results: The c-Met expression level in the control group (26.53 ± 3.50, 19.43–32.73) was significantly lower than in the infertile group (27.95 ± 2.86, 21.58–33.07) (p = 0.039). HGF expression was also significantly lower in the control group (3.25 ± 1.76, 0.83–8.25) than in the infertile group (4.87 ± 4.11, 1.01–21.58) (p = 0.043). In the overall group, c-Met expression showed a positive correlation with sperm motility percentage and a negative correlation with sperm count and sperm morphology percentage. Conclusions: The c-Met receptor and its ligand, hepatocyte growth factor, appear to be fundamental regulators of spermatogenesis, sperm motility and fertilization capacity.

Mechanisms of antifibrotic action of placental multipotent mesenchymal stromal cells

The aim of the study was to investigate the mechanisms of action of multipotent mesenchymal stromal cells (MMSCs) in a model of tetrachloromethane-induced liver fibrosis in mice. The experiments were performed on 50 mice. MSCs isolated from mouse placenta choriona were used in the study. MSCs were transplanted at a dose of 1 million cells into the tail vein. α-SMA, MMP-9, MMP 13, and TIMP-1 positive regions were analyzed by immunohistochemical method to evaluate the mechanisms of MMSCs action. Sirius red dye was used for connective tissue staining. HGF and TGF-β were determined by immunoassay in liver homogenate. We obtained that transplantation of MMSCs leads to a decrease in the area of α-SMA, TIMP-1 positive regions, an increase in the number of cells expressing MMP-9 and MMP 13. An increase in the level of hepatocyte growth factor and a decrease in transforming growth factor beta in liver homogenate after hMSCs transplantation in liver fibrosis was found. The results of the study demonstrated the efficacy of placental MMSCs in liver fibrosis. The antifibrotic mechanisms of action of hMSCs can include the ability to increase the expression of HGF, decrease the level of TGF-β, increase the production of matrix metalloproteinases-9 and -13, as well as the ability to inhibit the expression of tissue inhibitor of matrix metalloproteinases type 1.

The Effects of Amniotic Membrane Transplantation on Vocal Fold Regeneration.

Vocal fold (VF) scar and sulcus cause severe vocal problems, but optimal methods have not been established. Total replacement of the mucosa is required particularly for cases in which the whole lamina propria is occupied by severe fibrosis and vibratory function is totally lost. The amniotic membrane (AM) has been proven to have regenerative potential, as it contains stem cells and growth factors. The current study investigated the biocompatibility and effects of AM for regeneration of the VF mucosa. In vitro and in vivo studies. Vocal fold fibroblasts (VFFs) from 13 Sprague-Dawley rats were seeded on AM and subjected to histology and immunohistochemistry, and gene expressions in the VFFs on AM were examined in in vitro study. Twelve New Zealand White rabbits were used in in vivo study. VFs were stripped down and were reconstructed with AM. The regenerative effects were examined 3months later by histological examination. In vitro study indicated VFFs survived on AM and stained positively for Ki67, vimentin, and fibronectin. Gene expressions of Has1, Has2, and Hgf were significantly increased in the VFFs on AM compared with the other groups. The in vivo study indicated AM-transplanted VFs showed a significantly higher density of hyaluronic acid and lower density of collagen compared with sham VFs. The current preliminary study suggests biocompatibility and possible regenerative effects of AM for VFs. NA Laryngoscope, 132:2017-2025, 2022.

Elevated level of microRNA-210 at the initiation of muscular regeneration in acetic acid-induced non-ischemic skeletal muscular injury in mice.

The alteration in microRNA-210 level, a hypoxia-inducible microRNA, is not well known in non-ischemic tissue injury. In this study, we characterized the histopathological time course of acetic acid-induced skeletal muscle injury as a non-ischemic tissue injury model and investigated the expression of microRNA-210, hypoxia-inducible factor 1α, and growth factors using quantitative polymerase chain reaction analysis. After a single intramuscular dose of 3% (v/v) acetic acid to C57BL/6J mice, focal coagulative necrosis of muscle fibers was noted from 3 h after dosing and infiltration of F4/80 and Galectin-3 positive M2 macrophage was noted at 1 d after dosing. Muscular regeneration was initiated from 3 d, when M2 macrophage infiltration was most prominent, till 14 d after dosing. Hif1α and Hgf expression increased from 3 h onwards, and microRNA-210 level increased after 3 d after the treatment. However, no clear elevation in the levels of Igf1 or Vegf was observed. The infiltrative macrophages and regenerative muscle fibers were positive for hypoxia-inducible factor 1α, microRNA-210, and hepatocyte growth factor as assessed by immunohistochemistry or in situ hybridization. In this study, dominant infiltration of M2 macrophages at muscular necrosis and subsequent regeneration after a single intramuscular injection of acetic acid in mice were observed. The increase in hif1α level was observed just after the muscular injury in this non-ischemic tissue injury model, and the elevation in microRNA-210 level was noted at the initiation of tissue regeneration, indicating its effects on tissue protection and repair.

Abstract 10495: Mechanism of Cardiac Repair in Rat Myocardial Infarction Model Treated with Extracellular Vesicles from Differentiating Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: A few reports showed the therapeutic effects of extracellular vesicles (EVs) secreted from induced pluripotent stem cell-derived cardiomyocytes (iPSCM) on animal ischemic cardiomyopathy models. However, the mechanism of the cardiac function restoration after EVs administration remained unclear. Then, this study aimed to identify the mechanism for the restoration of cardiac function after iPSCM-EVs treatment. Methods: We collected EVs from differentiating iPSCM (TnT positive > 80%) culture medium. Comprehensive miRNA-seq and proteome analysis in the EVs were performed. In vitro myocardial contraction assay and wound healing assay, and ex vivo aortic ring assay were performed. In vivo , we designed a rat model of myocardial infarction by left anterior descending artery (LAD) ligation and divided rats into two groups. Two weeks after LAD ligation, iPSCM-EVs (Group EV) or PBS (Group Ctl) were injected peri-infarct zone. We analyzed the sequential cardiac function, histological analysis, and comprehensive RNA sequence analysis of the border zone before the treatment and 3, 7, 14, and 28 days after the treatment (n=5 at each time point in each group). Results: EVs showed the potential of increased myocyte contraction velocity (11±0.7 vs 6.6±0.6 μm/s, mean±SD, p < 0.01) and enhanced endothelial migration in vitro and facilitated angiogenesis (46±6 vs 13±5 sprouting count, p=0.02) ex vivo . In vivo , Group EVs showed higher LVEF (57±5 vs 38±4 % at day28, p < 0.01) and smaller LVDd (6.8±0.3 vs 8.5±0.8 mm at day28, p < 0.01). Histological analysis showed a lower % fibrosis area in Group EV (22±6 vs 12±5 % at day28, p < 0.01) and significantly higher microvascular (vWF positive: 1.7 at day14 and 2.1 times higher at day28) and arterial (vWF and αSMA positive: 4.3 at day14 and 6.2 times higher at day28) density. RNA expression analysis showed the expression of HGF, SDF-1, and IGF-1 increased in Group EVs on day3 and 7. CTGF and collagen 1 increased in Group Ctl on day14 and 28. Conclusions: Intracardiac injection of iPSCM-EVs improved cardiac function in rat MI model via angiogenesis, increased myocyte contraction, and reduced fibrosis. Comprehensive expression analysis suggested the mechanism of action of EVs in cardiac repair.

Abstract PO-113: The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support pancreatic cancer

Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. PIN1 overexpression in cancer contributes to cancer cell-intrinsic phenotypes including cellular proliferation and migration. While its pro-tumor functions have generated interest in therapeutic targeting of PIN1 for cancer treatment, the effects of PIN1 inhibition on tumor-associated stromal phenotypes have not yet been studied. We assessed pancreatic cancer xenografts and genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice that were treated with small molecule PIN1 inhibitors or crossed into a full body PIN1 knockout (Pin1−/−), and found that PIN1 inhibition or loss decreased tumor growth and extended overall survival. To interrogate a direct role for PIN1 in the stroma, we orthotopically injected a KPC cell line into syngeneic Pin1+/+ or Pin1−/− hosts and found dramatic reduction of tumor cell growth in Pin1−/− hosts. Further analysis of the Pin1−/− tumor microenvironment revealed decreased expression of alpha-SMA, a marker of myofibroblastic cancer associated fibroblasts (myCAFs), as well as decreased ECM deposition and/or organization. Pancreatic stellate cells (PSCs) activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. We, therefore, interrogated the role of PIN1 in PSCs. We found that loss of PIN1 in PSCs inhibits TGF-beta-induced stellate cell activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive changes to chromatin accessibility are impaired in the absence of PIN1, and suggests that specific transcription factor families may play a role in the PIN1-dependent response to TGF-beta. Further analysis of PSCs or CAFs with PIN1 loss indicated that, at baseline, these cells express gene programs consistent with the recently described antigen presenting CAFs (apCAFs). Finally, in addition to changes in cellular state and plasticity, we found that loss of PIN1 alters PSC secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of VEGF, resulting in altered cancer cell and vascular phenotypes. This work establishes a role for PIN1 in regulating fibroblast function and suggests that targeting PIN1 in cancer will have a broad anti-tumor effect. Our ongoing work continues to use 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the precise mechanisms by which PIN1 controls fibroblast phenotypes and impact of these changes on tumor phenotypes and outcomes. Citation Format: Ellen M. Langer, Isabel A. English, Vidhi Shah, Kevin MacPherson, Kayleigh M. Kresse, Brittany L. Allen-Petersen, Colin J. Daniel, Mara H. Sherman, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-113.

Three subtypes of lung cancer fibroblasts define distinct therapeutic paradigms.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Identification of a Three-Gene Signature Based on Epithelial-Mesenchymal Transition of Lung Adenocarcinoma Through Construction and Validation of a Risk-Prediction Model.

Epithelial-mesenchymal transition (EMT) process, which is regulated by genes of inducible factors and transcription factor family of signaling pathways, transforms epithelial cells into mesenchymal cells and is involved in tumor invasion and progression and increases tumor tolerance to clinical interventions. This study constructed a multigene marker for lung predicting the prognosis of lung adenocarcinoma (LUAD) patients by bioinformatic analysis based on EMT-related genes. Gene sets associated with EMT were downloaded from the EMT-gene database, and RNA-seq of LUAD and clinical information of patients were downloaded from the TCGA database. Differentially expressed genes were screened by difference analysis. Survival analysis was performed to identify genes associated with LUAD prognosis, and overlapping genes were taken for all the three. Prognosis-related genes were further determined by combining LASSO regression analysis for establishing a prediction signature, and the risk score equation for the prognostic model was established using multifactorial COX regression analysis to construct a survival prognostic model. The model accuracy was evaluated using subject working characteristic curves. According to the median value of risk score, samples were divided into a high-risk group and low-risk group to observe the correlation with the clinicopathological characteristics of patients. Combined with the results of one-way COX regression analysis, HGF, PTX3, and S100P were considered as independent predictors of LUAD prognosis. In lung cancer tissues, HGF and PTX3 expression was downregulated and S100P expression was upregulated. Kaplan-Meier, COX regression analysis showed that HGF, PTX3, and S100P were prognostic independent predictors of LUAD, and high expressions of all the three were all significantly associated with immune cell infiltration. The present study provided potential prognostic predictive biological markers for LUAD patients, and confirmed EMT as a key mechanism in LUAD progression.

Immunohistochemical expression of HGF and cMET in meningiomas

Abstract Background Meningiomas are the most common intracranial neoplasm together with gliomas; but unlike gliomas they are still graded according to their histopathological picture. Many growth factors and tyrosine kinase receptors have been studied in meningiomas in an attempt for prognostic stratification of patients, also to find a suitable targeted therapy for meningiomas. Aim of the study To correlate the immunohistochemical expression of HGF and cMET with the different WHO grades of meningioma in addition to brain invasion. Materials and methods This study included 60 cases of meningiomas (WHO grade I, n = 30 and WHO grade II & III, n = 30), brain invasion was present in 14 cases. Results HGF, cMET & HGF/cMET co-expression were positive in 60.0%, 65.0% and 58.3% respectively of the cases; WHO grade I meningioma showed positivity in 30.0%, 33.3% and 30.0% of the cases respectively in contrast with 90.0%, 96.7% and 86.7% of the cases of WHO grades II & III meningiomas respectively. The relation was statistically highly significant (p- value: <0.001). As regards cases with brain invasion the expression of HGF, cMET and HGF/cMET co-expression was found in 92.9%, 100.0% and 92.9% respectively in comparison with 50.0%, 54.3% and 47.8% in the rest of the cases of meningiomas respectively; the relation was statistically significant (p- value: 0.004). Conclusion The results suggest that HGF/cMET pathway may play a role in the progression of meningiomas from WHO grade I to higher grades II & III.

Overexpressed HGF promotes metastasis of squamous cell carcinoma of the head and neck through the PI3K/Akt and JNK signaling pathways.

Background: The role of HGF in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Methods: Reverse transcription PCR, western blotting, gelatin zymography, immunohistochemistry, actin polymerization, chemotaxis and migration assays were used in the authors' study. Results: HGF expression level was upregulated in SCCHN cells, which was associated with clinical stage; tumor, node, metastasis classification; and lymphatic invasion. SCCHN cells with high Met expression were sensitive to cell invasion, which was blocked by inhibiting PI3K/Akt and JNK. HGF induced MMP9 expression and enhanced its activity. Akt induced the activation of JNK through the PI3K/Akt and JNK signaling pathways. Conclusion: HGF upregulates MMP9 through the activation of the PI3K/Akt and JNK signaling pathways in SCCHN cells.

Periodontitis is associated to increased systemic inflammation in postmyocardial infarction patients

ObjectivePeriodontitis has been independently associated to cardiovascular disease. However, the biological mechanisms underlying such association are still partially unknown. Thus, this study aimed to discover immunological clues accounting for the...

Effect of Internal Iliac Artery Embolization Combined With Arterial Infusion Chemotherapy On Serum VEGF, HGF and IL-6 in Advanced Cervical Cancer

This study aimed to investigate the effect of internal iliac artery embolization combined with arterial infusion chemotherapy on serum VEGF, HGF and IL-6 in advanced cervical cancer. A total of 120 cases of patients with advanced cervical cancer and admitted to Binzhou Center Hospital from January 2014 to March 2016 were selected as research subjects. Among them, 59 patients received arterial infusion chemotherapy were enrolled in the control group, and 61 patients received arterial infusion chemotherapy combined with internal iliac artery embolization were included in the observation group. Treatment efficacy, serum VEGF, HGF and IL-6 expression, KPS score and QOL score, incidence of adverse reactions and 3-year survival rate of the two groups were recorded and compared. The result showed that the therapeutic effect of the observation group was significantly higher than that of the control group (P<0.05). The expression of serum VEGF, HGF and IL-6, KPS score and QOL score in the two groups were significantly improved after treatment, with more significant improvement in the observation group (P<0.05). There were no significant differences in the incidence of adverse reactions between the two groups (P>0.05). And the 3-year survival rate of the observation group was significantly higher than that of the control group (P<0.05). In conclusion, arterial infusion chemotherapy combined with internal iliac artery embolization has a better effect than arterial infusion chemotherapy alone in treating patients with advanced cervical cancer. It can effectively prolong the survival of patients, along with good safety, which is worthy of clinical promotion

A drug screen with approved compounds identifies amlexanox as a novel Wnt/β-catenin activator inducing lung epithelial organoid formation.

Background and Purpose:Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β-catenin signalling and assess its potential to activate lung epithelial cells and repair.Experimental Approach:We screened 1216 human-approved compounds for Wnt/β-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model.Key Results:The primary screen identified 16 compounds that significantly induced Wnt/β-catenin-dependent luciferase activity. Selected compounds activated Wnt/β-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β-catenin inhibition, confirming the Wnt/β-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces proregenerative signalling in emphysema.Conclusion and Implications:Using a drug screen based on Wnt/β-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.

Transcriptional Profile of Cytokines, Regulatory Mediators and TLR in Mesenchymal Stromal Cells after Inflammatory Signaling and Cell-Passaging.

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1β, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFβ. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.

3D-printed nerve guidance conduits multi-functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats

BackgroundNerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration.Methods3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve.ResultsThe inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair.Conclusions3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

The adipose-derived mesenchymal stem cell secretome promotes hepatic regeneration in miniature pigs after liver ischaemia-reperfusion combined with partial resection

BackgroundHepatic ischaemia-reperfusion injury (HIRI) is inevitable in complicated liver surgery and is a major factor leading to postoperative complications and liver dysfunction. Studies have shown that the paracrine mechanisms of stem cell may be essential to tissue repair and functional improvement after transplantation. However, the role of the adipose-derived mesenchymal stem cell secretome (ASC-secretome) in liver regeneration in large animals remains to be determined.MethodsTwenty-four miniature pigs were subjected to laparoscopic liver ischaemia-reperfusion combined with partial hepatectomy and divided into the following four groups: the saline group, the DMEM group, the ASC group and the ASC-secretome group. Serum and liver tissue samples were collected before the operation and at 1, 3 and 7 days after the operation, and changes in tissue pathology, serum inflammation, liver function, angiogenesis-related factors and liver tissue regeneration-related genes and proteins were evaluated.ResultsDetailed histological analysis showed that ASCs and the ASC-secretome changed pathological damage to liver tissue after liver ischaemia-reperfusion combined with partial hepatectomy (1 and 3 days: p < 0.01). Compared with the saline and DMEM control groups, the ASC-secretome group had significantly reduced expression levels of ALP (1 and 3 days: p < 0.05), ALT (1 day: p < 0.01; 3 days: p < 0.05) and AST (1 and 3 days: p < 0.01), which promoted the recovery of liver function. Moreover, detection of the expression levels of TNF-α and IL-1β (1 day: p < 0.01; 3 days: p < 0.05), IL-6 (1 and 3 days: p < 0.05) and IL-10 (1 and 3 days: p < 0.01) in serum confirmed that the ASC-secretome had obvious anti-inflammatory effects. In addition, the ASC-secretome increased the expression levels of ANG-1 (3 days: p < 0.01), ANG-2 (3 and 7 days: p < 0.01) and VEGF (1 and 7 days: p < 0.05; 3 days: p < 0.01) and promoted angiogenesis during liver regeneration. Moreover, it promoted the mRNA expression of HGF and Cyclin D1 (1 and 3 days: p < 0.01); increased the levels of p-STAT3 (1 and 3 days: p < 0.01), PCNA and Ki67 (1 and 3 days: p < 0.01; 7 days: p < 0.05); inhibited the negative feedback of SOCS3 (1 and 3 days: p < 0.01); and decreased the mRNA expression of TGF-β (3 days: p < 0.01). The cytokines and growth factors detected in the ASC-secretome included TNF-α, IL-6, IL-1β, ANG-1, ANG-2, VEGF and b-FGF.ConclusionThe ASC-secretome alleviates the inflammatory response induced by ischaemia-reperfusion combined with partial hepatectomy in miniature pigs and promotes liver regeneration.

The role of c-Met and VEGFR2 in glioblastoma resistance to bevacizumab

Dismal prognosis of glioblastoma (GBM) prompts for the identification of response predictors and therapeutic resistance mechanisms of current therapies. The authors investigated the impact of c-Met, HGF, VEGFR2 expression and microvessel density (MVD) in GBM patients submitted to second-line chemotherapy with bevacizumab. Immunohistochemical expression of c-Met, HGF, VEGFR2, and MVD was assessed in tumor specimens of GBM patients treated with bevacizumab, after progression under temozolomide. Survival analysis was evaluated according to the expression of the aforementioned biomarkers. c-Met overexpression was associated with a time-to-progression (TTP) after bevacizumab of 3 months (95% CI, 1.5–4.5) compared with a TTP of 7 months (95% CI, 4.6–9.4) in patients with low or no expression of c-Met (p = 0.05). VEGFR2 expression was associated with a TTP after bevacizumab of 3 months (95% CI, 1.8–4.2) compared with a TTP of 7 months (95% CI, 5.7–8.3) in patients with no tumoral expression of VEGFR2 (p = 0.009). Concomitant c-Met/VEGFR2 overexpression was associated with worse overall survival (13 months) compared with concomitant c-Met/VEGFR2 negative expression (19 months; p = 0.025). Our data support the hypothesis that c-Met and VEGFR2 overexpression have a role in the development of glioblastoma early resistance and might predict poorer responses to anti-angiogenic therapies.

Abstract LT012: The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer

Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. Due to its role in activation and stabilization of many oncogenes, we hypothesized that targeting PIN1 in pancreatic ductal adenocarcinoma (PDA) would slow tumor growth. We tested this hypothesis in vitro and in vivo with PIN1 inhibitors and/or genetic model systems. Pancreatic cancer cell lines knocked down for PIN1 or treated with PIN1 inhibitors showed decreased proliferation, invasion, and anchorage independent growth compared to control lines. Consistent with these in vitro results, treatment of pancreatic cancer xenografts or genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice with PIN1 inhibitors decreased tumor growth and extended overall survival. Similar results were seen in KPC mice that were crossed into a full body PIN1 knockout (PIN1−/−). Further analysis of KPC PIN1−/− tumors revealed not only reduced size of pancreatic tumors, but also decreased alpha-SMA expression and decreased ECM deposition in the stroma surrounding the tumors. PDA is characterized by a dense, desmoplastic tumor stroma that contributes to tumor growth, metastasis, and therapeutic resistance. Pancreatic stellate cells (PSCs) that are activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. To interrogate a direct role for PIN1 in the stroma, we first orthotopically injected a KPC cell line into syngeneic PIN1+/+ or PIN1−/− mice and found dramatic reduction of tumor cell growth in PIN1−/− hosts. Next, we analyzed PSCs in vitro and found that loss of PIN1 reduces their proliferation and alters their secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of SPINT1 and SPINT2, inhibitors of HGF activation. Conditioned media from control PSCs, but not from PSCs lacking PIN1 expression, activates the MET receptor on cancer cell lines, resulting in altered cancer cell phenotypes. In addition, we show that loss of PIN1 in PSCs inhibits TGF-beta induced stellate cells activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive chromatin changes are impaired in the absence of PIN1. Our ongoing work utilizes 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the mechanisms by which fibroblast phenotypes and the tumor-stromal crosstalk is impacted by PIN1. Citation Format: Ellen M. Langer, Isabel A. English, Kayleigh M. Kresse, Kevin MacPherson, Brittany L. Allen-Petersen, Colin J. Daniel, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT012.