Expression Of Hepatocyte Growth Factor Research Articles

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells.

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.

HGF-mediated crosstalk between cancer-associated fibroblasts and MET-unamplified gastric cancer cells activates coordinated tumorigenesis and metastasis

Cancer-associated fibroblasts (CAFs) are important components of tumor stroma and play a key role in tumor progression. CAFs involve in crosstalk with tumor cells through various kinds of cytokines. In the present study, we screened hepatocyte growth factor (HGF) as a cytokine predominantly originating from CAFs. CAFs-derived HGF was found to promote MET-unamplified gastric cancer (GC) proliferation, migration, and invasion through the activation of HGF/c-Met/STAT3/twist1 pathway. It also activated interleukin (IL)-6/IL-6R/JAK2/STAT3/twist1 pathway by up-regulating IL-6R expression. As IL-6 was also found to upregulate c-Met expression, we identified the cooperation of HGF and IL-6 in enhancing the characteristics of CAFs. In vivo experiments revealed that CAFs-derived HGF promoted tumorigenesis and metastasis of MET-unamplified GC. Gene set enrichment analysis (GSEA) was performed to confirm our findings. Our study found that the increased expression of HGF in CAFs induced by MET-unamplified GC contributed to the malignant phenotype of both MET-unamplified GC and CAFs in tumor microenvironment.

Mechanism of Chinese herbal formula QHF against breast cancer MCF-7 cells invasion and migration

ObjectiveTo investigate the effects of Chinese herbal formula Qinghuofu (QHF) on the migration and invasion of breast cancer MCF-7 cells and its possible molecular mechanisms, thereby providing a theoretical basis to find effective anti-cancer medicine and therapeutic targets for the treatment of anti-migration and anti-invasion of breast cancer. MethodsBreast cancer MCF-7 cells were treated with different QHF and other different reagents, CCK8 assay was used to detect the influence of the reagents on the proliferation of MCF-7 cells; Scrape migration and Transwell assay were used to quantitatively determine the migration and invasion effects of QHF and hepatocyte growth factor (HGF) on the MCF-7 cells. Subsequently, the c-Met inhibitor and its downstream ERK and PI3K inhibitors were used to investigate the relationship between the migration and invasion of MCF-7 cells, as well as its downstream MAPK/ERK and PI3K/Akt signaling pathways. The expression levels of HGF, c-Met, ERK, p-Akt, p-c-Met, p-ERK, p-Akt, MMP2, MMP9, and VEGF in breast cancer MCF-7 cells treated with QHF and other reagents were also examined. ResultsThe result indicated that formula QHF not only significantly inhibited the proliferation of MCF-7 cells, but also significantly suppressed the effects of HGF (40 ng/mL) on the proliferation and movement of MCF-7 cells, reducing the ability of the cells to invade and migrate. Western blot analysis indicated that QHF and c-Met inhibitor significantly decreased the expression of p-c-Met, p-ERK1, p-ERK2, p-Akt, MMP-2, MMP-9, and VEGF, while HGF significantly increased the expression of p-c-Met in MCF-7 cells; c-Met downstream ERK and PI3K inhibitors also significantly decreased the expression of MMP-2, MMP-9, and VEGF in MCF-7 cells; But the difference among c-Met, PI3K, ERK, and QHF group were not statistically significant. ConclusionQHF can prevent the proliferation, migration, and invasion of MCF-7 cells by inhibiting the HGF/c-Met and its downstream PI3K/Akt and MAPK/ERK signaling pathways; Thereby down-regulating the expression of HGF, p-Met, p-ERK1, p-ERK2, p-Akt, MMP-2, MMP-9, and VEGF.

C-Fos is necessary for HGF-mediated gene regulation and cell migration in Schwann cells

We previously reported that the expression of hepatocyte growth factor (HGF) was highly induced after peripheral nerve damage, and that c-Fos is one of many cellular genes whose expressions are affected by the increased level of HGF[1]. c-Fos is an important component of AP-1 heterodimer, but its role has not been clearly understood in the context of HGF and Schwann cells (SCs). In this study, we investigated the relationship between HGF and c-Fos. First, it was confirmed that the c-Fos was increased in SCs after nerve injury, while this effect abrogated by PHA-665752, an inhibitor of c-met receptor. When primary SCs were treated with recombinant HGF protein, c-Fos expression was regulated in a typical quick, transient fashion at both RNA and proteins levels. HGF-mediated induction of c-Fos expression was highly suppressed by specific inhibitors of ERK and CREB, respectively. The knock down of c-Fos expression by siRNA almost completely blocked various HGF-mediated effects in SCs, such as induction of gene expression of GDNF, LIF, and c-Myc, and migration of SCs, indicating that c-Fos might play a key role in HGF effects. Taken together, our results suggested that c-Fos plays a key role(s) in HGF-mediated effects on neurotrophic genes and cell migration.

C-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors.

Type II testicular germ cell tumors (TGCTs) represent the most frequent malignancy in Caucasian males (20–40 years). Even if diagnosed with disseminated disease, >80% of patients are cured; however, a small percentage of cases progress and result in death. It is commonly accepted that these cancers arise from a disturbed testicular embryonic niche that leads to the block of gonocyte differentiation. The subsequent development of the invasive seminomas and non-seminomas is due to a combination of genetic, epigenetic and microenvironment-based alterations (genvironment). Hepatocyte growth factor (HGF) is present in the testicular microenvironment, together with its receptor c-MET, from early embryonic development to an adult stage. In addition, c-MET is a well-known proto-oncogene involved in the onset and progression of various human cancers. Herein, we have investigated the expression and availability of HGF and c-MET in TCam-2, NCCIT and NT2D1 cells, which are type II (T)GCT representative cell lines, and the effect of c-MET activation/repression on the regulation of cancerous biological processes. We found that NT2D1 cells increase their proliferation, polarized migration, and invasion in response to HGF administration. NCCIT cells respond to HGF stimulation only partially, whereas TCam-2 cells do not respond to HGF, at least according to the investigated parameters. Interestingly, the immunohistochemical study of c-MET distribution in TGCTs confirm its presence in both seminoma and non-seminoma lesions with different patterns. Notably, we found the highest c-MET immunoreactivity in the epithelial elements of the various components of TGCTs: teratoma, yolk sac tumor and choriocarcinoma.

Abstract 330: Systemic Blocking Exosome Formation/Release Improves Ischemic Hindlimb Repair in Diabetic db/db Mice

Background: Recent studies demonstrated that plasma exosomes from diabetic patients and animals lose their ability to maintain physiological properties of cardiomyocytes. Exosome inhibitor GW 4869 (GW) tended to improve heart function in diabetic mice with myocardial infarction. We tested the hypotheses that systemic blocking exosome synthesis/formation improves ischemic hindlimb repair in diabetes. Methods and Results: Unilateral hindlimb ischemia (HLI) surgery was conducted by ligation of left femoral artery in 12-week male db/db and db/+ mice which were administrated with either GW (2 mg/kg body weight, i.p) or vehicle for 4 weeks starting from one week before HLI. Blood perfusion in ischemic hindlimbs was examined by Laser Doppler at pre-HLI, and 0, 3, 7, 14 and 21 days post-HLI. Plasma exosomes were isolated by standard ultracentrifugation method and counted by NanoSight. GW increased blood concentration of CD31 + /Sca-1 + cell 3-day post-HLI and decreased necrosis and loss of toe/toenail; improved blood flow; enhanced capillary/arterial density determined by CD31 and SMA-α staining and decreased fibrosis in the muscles of ischemic hindlimbs in db/db mice 21-day post-HLI. Plasma exosomes from db/db mice significantly impaired human cardiac microvascular endothelial cells (HCMVECs) tube formation and migration. Mechanistically, hepatocyte growth factor (HGF) expression was decreased in bone marrow-derived endothelial progenitor cells (EPCs) from db/db mice and HCMVECs treated with diabetic plasma exosomes. Administration of GW increased HGF level in diabetic EPCs. Furthermore, diabetic plasma exosomes decreased H3K4me3 (active maker of transcription) whereas increased H3K27me3 (suppressive marker of transcription) at HGF promoter. Finally, treatment of HCMEVs with exogenous HGF rescued diabetic exosomes impaired tube formation of HCMVEC. Conclusions: Systemic inhibition of exosome synthesis/formation by GW increased ischemic limb repair in diabetic db/db mice, at least partially, via histone methylation mediated suppression of HGF. Our findings suggest that therapeutic targeting of systemic dysfunctional exosomes could represent a new avenue for therapeutics of ischemic tissue injury in patients with diabetes.

Hepatocyte Growth Factor Regulates the miR-206-HDAC4 Cascade to Control Neurogenic Muscle Atrophy following Surgical Denervation in Mice

Hepatocyte growth factor (HGF) has been well characterized for its roles in the migration of muscle progenitors during embryogenesis and the differentiation of muscle stem cells, but its function in adult neurogenic muscle atrophic conditions is poorly understood. Here we investigated whether HGF/c-met signaling has any effects on muscle-atrophic conditions. It was found that HGF expression was upregulated in skeletal muscle tissue following surgical denervation and in hSOD1-G93A transgenic mice showing severe muscle loss. Pharmacological inhibition of the c-met receptor decreased the expression level of pri-miR-206, enhanced that of HDAC4 and atrogenes, and resulted in increased muscle atrophy. In C2C12 cells, HGF inhibited phosphorylation of Smad3 and relieved TGF-β-mediated suppression of miR-206 expression via JNK. When extra HGF was exogenously provided through intramuscular injection of plasmid DNA expressing HGF, the extent of muscle atrophy was reduced, and the levels of all affected biochemical markers were changed accordingly, including those of primary and mature miR-206, HDAC4, and various atrogenes. Taken together, our finding suggested that HGF might play an important role in regard to neurogenic muscle atrophy and that HGF might be used as a platform to develop therapeutic agents for neuromuscular disorders.

Effective Amelioration of Liver Fibrosis Through Lentiviral Vector Carrying Toxoplasma gondii gra15II in Murine Model.

Our previous investigations indicated that in vitro polarization of mouse macrophages by Toxoplasma gondii type II strain dense granule protein 15 (GRA15II), one of the genotype-associated effectors of T. gondii, induced the phenotypes of classically activated macrophage (M1). Transfusion of the cells to mice may effectively alleviated hepatic fibrosis caused by schistosomiasis. The purpose of the study was to identify whether liver macrophages can be in vivo driven to M1 macrophages by lentiviral vector (LV) carrying GRA15II gene (LV-gra15II) and to explore the potential mechanism by which the LV-gra15II-activated liver macrophage (LV-gra15II-M) ameliorates the hepatic fibrosis in schistosomiasis. The mice were treated with LV-gra15II by hydrodynamic injection via the tail vein followed by challenge of Schistosoma japonicum (S. japonicum). Our experiments showed that LV-gra15II was successfully delivered to liver macrophages and GRA15II was persistently expressed in the macrophages of mice for at least 2 months. Furthermore, the LV-gra15II infected macrophages were polarized to M1 macrophages in vivo. Consequently, mice with schistosomiasis receiving LV-gra15II injection displayed a remarkable amelioration of liver granuloma formation and collagen deposition in association with downregulated expression of transforming growth factor-beta1, arginase 1 (Arg-1), α-smooth muscle actin, and an increased expression of matrix metalloproteinase 13 (MMP13). Simultaneously, no negative effects of liver function and vitality of mice were noted. The in vitro experiments indicated that the C-C motif chemokine ligand 2 and nitric oxide level were elevated in LV-gra15II-M cultural supernatants; hepatocyte growth factor expression was enhanced in LV-gra15II-M. In addition, LV-gra15II-M not only secreted MMP13, which greatly degraded type I collagen, but also induced murine hepatic stellate cell (HSC) line (JS1) apoptosis in the co-culture system. Taken together, we identified for the first time that LV-gra15II may in vivo drive liver macrophages to M1 macrophage phenotypes, which helps for alteration of the liver fibrotic microenvironment with collagen dissolution, HSC deactivation, apoptosis and hepatocyte protection. Our study gives an insight into the use of gene delivery with parasite-derived immunomodulatory factor as a potential immune cell activating agent to re-equilibrate the other pathogen-induced immune response in some chronic diseases.

Identification of genomic and molecular traits that present therapeutic vulnerability to HGF-targeted therapy in glioblastoma

Cancer is a complex disease with profound genomic alterations and extensive heterogeneity. Recent studies on large-scale genomics have shed light on the impact of core oncogenic pathways, which are frequently dysregulated in a wide spectrum of cancer types. Aberrant activation of the hepatocyte growth factor (HGF) signaling axis has been associated with promoting various oncogenic programs during tumor initiation, progression, and treatment resistance. As a result, HGF-targeted therapy has emerged as an attractive therapeutic approach. However, recent clinical trials involving HGF-targeted therapies have demonstrated rather disappointing results. Thus, an alternative, in-depth assessment of new patient stratification is necessary to shift the current clinical course. To address such challenges, we have evaluated the therapeutic efficacy of YYB-101, an HGF-neutralizing antibody, in a series of primary glioblastoma stem cells (GSCs) both in vitro and in vivo. Furthermore, we performed genome and transcriptome analysis to determine genetic and molecular traits that exhibit therapeutic susceptibility to HGF-mediated therapy. We have identified several differentially expressed genes, including MET, KDR, and SOX3, which are associated with tumor invasiveness, malignancy, and unfavorable prognosis in glioblastoma patients. We also demonstrated the HGF-MET signaling axis as a key molecular determinant in GSC invasion, and we discovered that a significant association in HGF expression existed between mesenchymal phenotype and immune cell recruitment. Upregulation of MET and mesenchymal cellular state are essential in generating HGF-mediated therapeutic responses. Our results provide an important framework for evaluating HGF-targeted therapy in future clinical settings.

Pulmonary pericytes regulate lung morphogenesis

Blood vessels are essential for blood circulation but also control organ growth, homeostasis, and regeneration, which has been attributed to the release of paracrine signals by endothelial cells. Endothelial tubules are associated with specialised mesenchymal cells, termed pericytes, which help to maintain vessel wall integrity. Here we identify pericytes as regulators of epithelial and endothelial morphogenesis in postnatal lung. Mice lacking expression of the Hippo pathway components YAP and TAZ in pericytes show defective alveologenesis. Mutant pericytes are present in normal numbers but display strongly reduced expression of hepatocyte growth factor leading to impaired activation of the c-Met receptor, which is expressed by alveolar epithelial cells. YAP and TAZ are also required for expression of angiopoietin-1 by pulmonary pericytes, which also controls hepatocyte growth factor expression and thereby alveologenesis in an autocrine fashion. These findings establish that pericytes have important, organ-specific signalling properties and coordinate the behavior of epithelial and vascular cells during lung morphogenesis.

MiR-26a modulates HGF and STAT3 effects on the kidney repair process in a glycerol-induced AKI model in rats.

Acute kidney injury is mostly reversible, and hepatocyte growth factor (HGF) has a relevant role in the tissue repair. MicroRNA (miR)-26a is an endogenous modulator of HGF. The role of miR-26a in the kidney repair process was evaluated in Wistar rats submitted to an acute kidney injury model of rhabdomyolysis induced by glycerol (6 mL/kg). Animals were evaluated 3, 12, 48, 96, and 120 hours after glycerol injection. Serum creatinine (SCr) and gene expression of HGF, c-met, signal transducer and activator of transcription 3 (STAT3), and miR-26a were estimated. Also, tubular NK52E cells were transfected with anti-miR26a and stimulated with Fe3+ for 24 hours to mimic the effects of myoglobin in vitro. SCr was highest after 48 hours. After 96 hours, SCr started to decrease, characterizing the recovery phase, with normalization after 120 hours. HGF expression increased during the onset phase (3 hours), with a low relationship with miR-26a. In contrast, in the recovery phase, the increase in miR-26a was coincident with HGF messenger RNA suppression, suggesting that in the recovery phase, miR-26a may have a role in HGF modulation. Fe3+ induced cellular death after 3 hours and proliferation after 24 hours. There was no correlation between miR-26a and STAT3 during the death phase; however, during the proliferation phase, an increase in STAT3 was paralleled with a decrease in miR-26a. miR-26a silencing induced increases in cell viability and the phosphorylated form of STAT3 protein expression in cells receiving Fe3+ . In conclusion, miR-26a may have a key role in modulating HGF levels after its proliferative effects have been triggered.

Dysregulated HAI-2 Plays an Important Role in Renal Cell Carcinoma Bone Metastasis through Ligand-Dependent MET Phosphorylation.

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone metastasis. In this study, we employed a mouse model of renal cell carcinoma (RCC) bone metastasis to clarify the significance of the HGF/MET signaling axis and the regulator of HGF activator inhibitor type-2 (HAI-2). Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of mice to prepare the mouse model of bone metastasis. The formation of bone metastasis was confirmed by whole-body bioluminescent imaging, and specimens were extracted. Expression of HGF/MET-related molecules was analyzed. Based on the results, we produced HAI-2 stable knockdown 786-O cells, and analyzed invasiveness and motility. Expression of HGF and matriptase was increased in bone metastasis compared with the control, while that of HAI-2 was decreased. Furthermore, we confirmed increased phosphorylation of MET in bone metastasis. The expression of matriptase was upregulated, and both invasiveness and motility were increased significantly by knockdown of HAI-2. The significance of ligand-dependent MET activation in RCC bone metastasis is considered, and HAI-2 may be an important regulator in this system.

Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions

Aims: To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Methods: Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. Results: The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β₁) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β₁ at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. Conclusions: bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model.

Enriched Environment Elicits Proangiogenic Mechanisms After Focal Cerebral Ischemia

Brain has limited capacity for spontaneous recovery of lost function after stroke. Exposure to enriched environment (EE) can facilitate functional recovery, but mechanisms underlying this effect are poorly understood. Here, we used a middle cerebral artery occlusion (MCAO) model to investigate the impact of EE on angiogenesis in the post-ischemic brain in adult male Sprague Dawley rats, and examined whether blood-borne factors may contribute. Compared with standard cage (SC), exposure to EE was associated with greater improvement in neurological function, higher peri-infarct vascular density, and higher chronic post-ischemic cerebral blood flow assessed by laser speckle imaging. The effect persisted for at least 28days. EE also enhanced the expression of hepatocyte growth factor in the peri-ischemic cortex when measured 15days after MCAO. Interestingly, serum from rats exposed to EE after MCAO showed elevated levels of hepatocyte growth factor, and plasma or serum from rats exposed to EE after MCAO enhanced the survival and proliferation of cultured endothelial cells, in vitro, when compared with control plasma or serum from SC group after MCAO. Together, our data suggest that exposure to EE promotes angiogenesis in the ischemic brain that may in part be mediated by blood-borne factors.

Endothelial Notch activation reshapes the angiocrine of sinusoidal endothelia to aggravate liver fibrosis and blunt regeneration in mice.

Liver sinusoidal endothelial cells (LSECs) critically regulate liver homeostasis and diseases through angiocrine factors. Notch is critical in endothelial cells (ECs). In the current study, Notch signaling was activated by inducible EC‐specific expression of the Notch intracellular domain (NIC). We found that endothelial Notch activation damaged liver homeostasis. Notch activation resulted in decreased fenestration and increased basement membrane, and a gene expression profile with decreased LSEC‐associated genes and increased continuous EC‐associated genes, suggesting LSEC dedifferentiation. Consistently, endothelial Notch activation enhanced hepatic fibrosis (HF) induced by CCl4. Notch activation attenuated endothelial nitric oxide synthase (eNOS)/soluble guanylate cyclase (sGC) signaling, and activation of sGC by 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole (YC‐1) reversed the dedifferentiation phenotype. In addition, Notch activation subverted the hepatocyte‐supporting angiocrine profile of LSECs by down‐regulating critical hepatocyte mitogens, including Wnt2a, Wnt9b, and hepatocyte growth factor (HGF). This led to compromised hepatocyte proliferation under both quiescent and regenerating conditions. Whereas expression of Wnt2a and Wnt9b was dependent on eNOS‐sGC signaling, HGF expression was not rescued by the sGC activator, suggesting heterogeneous mechanisms of LSECs to maintain hepatocyte homeostasis. Conclusion: Endothelial Notch activation results in LSEC dedifferentiation and accelerated liver fibrogenesis through eNOS‐sGC signaling, and alters the angiocrine profile of LSECs to compromise hepatocyte proliferation and liver regeneration (LR). (Hepatology 2018).

Correction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model

[This corrects the article DOI: 10.1371/journal.pone.0175807.].

Effective control of neuropathic pain by transient expression of hepatocyte growth factor in a mouse chronic constriction injury model.

Hepatocyte growth factor (HGF) is a multifunctional protein that contains angiogenic and neurotrophic properties. In the current study, we investigated the analgesic effects of HGF by using a plasmid DNA that was designed to express 2 isoforms of human HGF—pCK-HGF-X7 (or VM202)—in a chronic constriction injury (CCI) –induced mouse neuropathic pain model. Intramuscular injection of pCK-HGF-X7 into proximal thigh muscle induced the expression of HGF in the muscle, sciatic nerve, and dorsal root ganglia (DRG). This gene transfer procedure significantly attenuated mechanical allodynia and thermal hyperalgesia after CCI. Injury-induced expression of activating transcription factor 3, calcium channel subunit α2δ1, and CSF1 in the ipsilateral DRG neurons was markedly down-regulated in the pCK-HGF-X7–treated group, which suggested that HGF might exert its analgesic effects by inhibiting pain-mediating genes in the sensory neurons. In addition, suppressed CSF1 expression in DRG neurons by pCK-HGF-X7 treatment was accompanied by a noticeable suppression of the nerve injury–induced glial cell activation in the spinal cord dorsal horn. Taken together, our data show that pCK-HGF-X7 attenuates nerve injury–induced neuropathic pain by inhibiting pain-related factors in DRG neurons and subsequent spinal cord glial activation, which suggests its therapeutic efficacy in the treatment of neuropathic pain.—Nho, B., Lee, J., Lee, J., Ko, K. R., Lee, S. J., Kim, S. Effective control of neuropathic pain by transient expression of hepatocyte growth factor in a mouse chronic constriction injury model.

Mechanism of tissue-engineered bone recruiting endogenous mesenchymal stem cells towards bone regeneration

Objective To investigate the mechanism of implanted tissue-engineered bone (TEB) recruiting endogenous mesenchymal stem cells (BMSCs) towards bone regeneration after traumatic bone defect. Methods In vivo experiments: 2 mm of diaphysis and periosteum were removed from the middle of the femoral shaft in 8 week old FVB/N mice to form a large segment of bone defect. Demineralized bone matrix (DBM) and TEB were implanted into the defect area and fixated. All mice were randomly divided into DBM group (n=18) and TEB group (n=18). The results were observed 24 hours after implantation: (1) flow cytometry was used to evaluate the number of mobilized host BMSCs into the blood; (2) non-invasive bioluminescent imaging was used to observe the ability of two groups in recruiting mouse bone marrow derived mesenchymal stem cells (mBMSCs) in peripheral blood to the defect area; (3) ELISA was used to evaluate the stromal cell-derived factor 1 (SDF-1) content in peripheral blood of two groups. In vitro experiments: (1) transwell assay was conducted to evaluate the ability of SDF-1 (100 ng/ml) in promoting the migration of human bone marrow derived mesenchymal stem cells (hBMSCs). SDF-1/C-X-C motif chemokine receptor-4 (CXCR4) pathway was blocked by the selective CXCR4 antagonist Plerixafor (AMD3100). The experimental groups were divided into control group, SDF-1 group, and SDF-1+ AMD3100 group. (2) The co-culture system of human umbilical vein endothelial cells (hUVECs) and hBMSCs was established, and cells were stimulated by SDF-1. The experimental groups were divided into hBMSCs group, hBMSCs+ hUVECs group, and hBMSCs+ hUVECs (AMD3100 pretreatment) group. Transwell assays were used to compare the migration of hBMSCs in each group. ELISA was used to detect the concentration of hepatocyte growth factor (HGF) in the co-culture supernatant. (3) In vitro cultured hUVECs were stimulated by SDF-1 and SDF-1/CXCR4 pathway was antagonized by AMD3100. The experimental groups were divided into control group, SDF-1 group, and SDF-1+ AMD3100 group. Quantitative real-time polymerase chain reaction (qRT PCR) was used to evaluate the expression of HGF in each group. Results In vivo experiments: 24 h after transplantation, the number of BMSCs and SDF-1 concentration in the TEB group were significantly higher than those in the DBM group (P 0.05); (3) qRT-PCR showed that the expression of HGF was significantly increased in the SDF-1 group compared with the control group (P<0.05). After antagonizing SDF-1/CXCR4, HGF expression in the SDF-1+ AMD3100 group was significantly lower than that in the SDF-1 group. Conclusions TEB transplantation in traumatic bone defect can significantly increase the concentration of chemokine SDF-1 in vivo and effectively promote the mobilization of endogenous MSCs and recruitment of circulating MSCs. SDF-1 not only directly promotes the migration of hBMSCs through SDF-1/CXCR4 pathway, but also up-regulates the expression and secretion of HGF in vascular cells to further amplify the chemotactic effect of SDF-1 on hBMSCs. Key words: Tissue engineering; Mesenchymal stem cells; Stromal cell-derived factor-1

Comparison of the regenerative cytokine profile of adipose-derived stromal cells (ASCs) harvested via abdominoplasty, suction-assisted liposuction (SAL) and ultrasound-assisted liposuction (UAL)

Mesenchymal stem cells are already used in numerous areas of medicine. In the field of plastic surgery, the main focus of recent research has been to utilise adipose-derived stromal cells (ASCs), mainly harvested via liposuction. The ability to release regenerative cytokines is supposed to be the key mechanism for the therapeutic efficacy of ASCs. There are currently a variety of methods and devices in clinical use for harvesting lipoaspirate and consequently ASCs. For most of these devices, the effect on the regenerative cytokine profile of ASCs is unknown. The aim of this study is to compare the expression of key regenerative cytokines of ASCs derived by three standard procedures of plastic surgery: abdominoplasty, suction-assisted liposuction (SAL) and ultrasound-assisted liposuction (UAL). UAL and SAL lipoaspirates were obtained from 3 healthy female volunteers before abdominoplasty was performed in the same patients. ASCs were isolated from UAL and SAL lipoaspirates as well as from abdominoplasty resections and RNA expression of important regenerative cytokines (HGF (hepatocyte growth factor), FGF-2 (basic fibroblast growth factor), MCP-1 (monocyte chemotactic protein 1), SDF-1 (stromal cell-derived factor 1) and VEGF (vascular endothelial growth factor) was compared. There was no difference in the expression of HGF, FGF-2, SDF-1 and VEGF between the individual samples, but we were able to demonstrate significantly increased expression of MCP-1 in ASCs obtained from UAL lipoaspirate. UAL and SAL lipoaspirates are suitable for obtaining functional ASCs and are comparable to minimally manipulated cells from abdominoplasty resections.

Keratin 19 Expression in Hepatocellular Carcinoma Is Regulated by Fibroblast-Derived HGF via a MET-ERK1/2-AP1 and SP1 Axis.

Keratin (KRT) 19 is a poor prognostic marker for hepatocellular carcinoma (HCC); however, regulatory mechanisms underlying its expression remain unclear. We have previously reported the presence of fibrous tumor stroma in KRT19-positive HCC, suggesting that cross-talk between cancer-associated fibroblasts (CAF) and tumor epithelial cells could regulate KRT19 expression. This was investigated in this study using an in vitro model of paracrine interaction between HCC cell lines (HepG2, SNU423) and hepatic stellate cells (HSC), a major source of hepatic myofibroblasts. HSCs upregulated transcription and translation of KRT19 in HCC cells via paracrine interactions. Mechanistically, hepatocyte growth factor (HGF) from HSCs activated c-MET and the MEK-ERK1/2 pathway, which upregulated KRT19 expression in HCC cells. Furthermore, AP1 (JUN/FOSL1) and SP1, downstream transcriptional activators of ERK1/2, activated KRT19 expression in HCC cells. In clinical specimens of human HCC (n = 339), HGF and KRT19 protein expression correlated with CAF levels. In addition, HGF or MET protein expression was associated with FOSL1 and KRT19 expression and was found to be a poor prognostic factor. Analysis of data from The Cancer Genome Atlas also revealed KRT19 expression was closely associated with CAF and MET-mediated signaling activities. These results provide insights into the molecular background of KRT19-positive HCC that display an aggressive phenotype.Significance: These findings reveal KRT19 expression in hepatocellular carcinoma is regulated by cross-talk between cancer-associated fibroblasts and HCC cells, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(7); 1619-31. ©2018 AACR.