Release Of Hepatocyte Growth Factor Research Articles

A Multifunctional Nanocomposite Hydrogel Delivery System Based on Dual-Loaded Liposomes for Scarless Wound Healing.

Increased inflammatory responses and oxidative stress at the wound site following skin trauma impair healing. Furthermore, skin scarring places fibroblasts under severe mechanical stress and aggravates pathological fibrosis. A novel liposomal composite hydrogel is engineered for wound microenvironment remodeling, incorporating dual-loaded liposomes into gelatin methacrylate to create a nanocomposite hydrogel. Notably, tetrahydrocurcumin (THC) and hepatocyte growth factor (HGF) are encapsulated in the hydrophobic and hydrophilic layers of liposomes, respectively. The composite hydrogel maintains porous nanoarchitecture, demonstrating sustainable THC and HGF release and enhanced mechanical properties and biocompatibility. This system effectively promotes cell proliferation and angiogenesis and attenuates apoptosis. It decreases the expression of the inflammatory factors by inhibiting the high-mobility group box /receptor for advanced glycation end product/NF-κB (HMGB1/RAGE/NF-κB)pathway and increases macrophage polarization from M1 to M2 in vitro, effectively controlling inflammatory responses. It exhibits remarkable antioxidant properties by scavenging excess reactive oxygen species and free radicals. Most importantly, it effectively prevents scar formation by restraining the transforming growth factor beta (TGF-β)/Smads pathway that downregulates associated fibrotic factors. It demonstrates strong therapeutic effects against inflammation and fibrosis in a rat skin wound model with biosafety, advancing the development of innovative hydrogel-based therapeutic delivery strategies for clinical scarless wound therapy.

Impact of Fibrin Gel Architecture on Hepatocyte Growth Factor Release and Its Role in Modulating Cell Behavior for Tissue Regeneration.

A novel scaffold design has been created to enhance tissue engineering and regenerative medicine by optimizing the controlled, prolonged release of Hepatocyte Growth Factor (HGF), a powerful chemoattractant for endogenous mesenchymal stem cells. We present a new stacked scaffold that is made up of three different fibrin gel layers, each of which has HGF integrated into the matrix. The design attempts to preserve HGF's regenerative properties for long periods of time, which is necessary for complex tissue regeneration. These multi-layered fibrin gels have been mechanically evaluated using rheometry, and their degradation behavior has been studied using D-Dimer ELISA. Understanding the kinetics of HGF release from this novel scaffold configuration is essential for understanding HGF's long-term sustained bioactivity. A range of cell-based tests were carried out to verify the functionality of HGF following extended incorporation. These tests included 2-photon microscopy using phalloidin staining to examine cellular morphology, SEM analysis for scaffold-cell interactions, and scratch and scatter assays to assess migration and motility. The analyses show that the novel stacking scaffold promotes vital cellular processes for tissue regeneration in addition to supporting HGF's bioactivity. This scaffold design was developed for in situ tissue engineering. Using the body as a bioreactor, the scaffold should recruit mesenchymal stem cells from their niche, thus combining the regenerative abilities of HGF and MSCs to promote tissue remodeling and wound repair.

GPR55 inhibits the pro-adipogenic activity of anandamide in human adipose stromal cells

The endocannabinoid anandamide (AEA) stimulates adipogenesis via the cannabinoid receptor CB1 in adipose stromal cells (ASCs). However, AEA interacts also with nonclassical cannabinoid receptors, including transient receptor potential cation channel (TRPV)1 and G protein-coupled receptor (GPR)55. Their roles in AEA mediated adipogenesis of human ASCs have not been investigated.We examined the receptor-expressions by immunostaining on human ASCs and tested their functionality by measuring the expression of immediate early genes (IEGs) related to the transcription factor-complex AP-1 upon exposition to receptor agonists. Cells were stimulated with increasing concentrations of specific ligands to investigate the effects on ASC viability (proliferation and metabolic activity), secretory activity, and AEA mediated differentiation.ASCs expressed both receptors, and their activation suppressed IEG expression. TRPV1 did not affect viability or cytokine secretion. GPR55 decreased proliferation, and it inhibited the release of hepatocyte growth factor. Blocking GPR55 increased the pro-adipogenic activity of AEA. These data suggest that GPR55 functions as negative regulator of cannabinoid mediated pro-adipogenic capacity in ASCs.

Understanding how MSCs reverse radiation-induced fibrosis: HGF vs. TGF-B1

Understanding how MSCs reverse radiation-induced fibrosis: HGF <i>vs.</i> TGF-B1

Development of heparinized and hepatocyte growth factor-coated acellular scaffolds using porcine carotid arteries.

Tissue-engineered blood vessel substitutes have been developed due to the lack of suitable small-diameter vascular grafts. Xenogeneic extracellular matrix (ECM) scaffolds have the potential to provide an ideal source for off-the-shelf vascular grafts. In this study, porcine carotid arteries were used to develop ECM scaffolds by decellularization and coating with heparin and hepatocyte growth factor (HGF). After decellularization, cellular and nucleic materials were successfully removed with preservation of the main compositions (collagen, elastin, and basement membrane) of the native ECM. The ultimate tensile strength, suture strength, and burst pressure were significantly increased after cross-linking. Pore size distribution analysis revealed a porous structure within ECM scaffolds with a high distribution of pores larger than 10 μm. Heparinized scaffolds exhibited sustained release of heparin in vitro and showed potent anticoagulant activity by prolonging activated partial thromboplastin time. The scaffolds showed an enhanced HGF binding capacity as well as a constant release of HGF as a result of heparin modification. When implanted subcutaneously in rats, the modified scaffolds revealed good biocompatibility with enzyme degradation resistance, mitigated immune response, and anti-calcification. In conclusion, heparinized and HGF-coated acellular porcine carotid arteries may be a promising biological scaffold for tissue-engineered vascular grafts.

Microenvironmental modulation in tandem with human stem cell transplantation enhances functional recovery after chronic complete spinal cord injury

While rapid advancements in regenerative medicine strategies for spinal cord injury (SCI) have been made, most research in this field has focused on the early stages of incomplete injury. However, the majority of patients experience chronic severe injury; therefore, treatments for these situations are fundamentally important. Here, we hypothesized that environmental modulation via a clinically relevant hepatocyte growth factor (HGF)-releasing scaffold and human iPS cell-derived neural stem/progenitor cells (hNS/PCs) transplantation contributes to functional recovery after chronic complete transection SCI. Effective release of HGF from a collagen scaffold induced progressive axonal elongation and increased grafted cell viability by activating microglia/macrophages and meningeal cells, inhibiting inflammation, reducing scar formation, and enhancing vascularization. Furthermore, hNS/PCs transplantation enhanced endogenous neuronal regrowth, the extension of graft axons, and the formation of circuits around the lesion and lumbar enlargement between host and graft neurons, resulting in the restoration of locomotor and urinary function. This study presents an effective therapeutic strategy for severe chronic SCI and provides evidence for the feasibility of regenerative medicine strategies using clinically relevant materials.

Abstract 13947: Heparin-Mediated Release of Hepatocyte Growth Factor at the Acute Phase of STEMI

Introduction: Hepatocyte Growth Factor (HGF) is a cytokine promoting cell survival via its receptor c-met. Heparin injection is known to induce a massive release of native HGF in blood. Heparin is used at the acute phase of myocardial infarction (MI). However, HGF release at the acute phase of MI and its potential cardioprotective effect is unknown. Hypothesis: We aimed to assess HGF kinetics in a cohort of ST-elevation myocardial infarction (STEMI) and its potential cardioprotective effect. Methods: We prospectively included consecutive STEMI patients admitted in our hospital from 2016 to 2019. All patients underwent coronary angiography with revascularization. Sera were collected at 5 time points (admission, 4 hours (H4), H24, H48 and 1 month after STEMI). Samples were stored at -80°C. HGF levels were assess by ELISA. We used a mouse model of myocardial ischemia reperfusion (ligation of the left anterior descending coronary artery followed by reperfusion) to evaluate HGF effect. Results: We included 230 patients. We observed an intense and early peak of HGF as early as admission, before revascularization (8750 pg/ml, IQR [8021-9492]) followed by a rapid decrease within the first 48h (p&lt;0.0001). We demonstrated that this rapid increase was induced by heparin administration (unfractionated heparin or low molecular weight heparins). To evaluate the effect of HGF on myocardial infarct size we administrated HGF or placebo in mice during myocardial reperfusion. In the HGF group, infarct size was significantly smaller compared to the control group (52.0% of left ventricle vs 58.0%, p=0.002). Conclusions: We demonstrated an intense and early HGF peak at the acute phase of STEMI, induced by heparin. In our preclinical mouse model, HGF had cardioprotective effect after MI. Thus we may suppose that heparin-mediated release of HGF at the acute phase of STEMI have cardioprotective effect that may confer a basal cardioprotection in all patients.

The Performance of Nonwoven PLLA Scaffolds of Different Thickness for Stem Cells Seeding and Implantation.

The 3D reconstruction of 100 μm- and 600 μm-thick fibrous poly-L/L-lactide scaffolds was performed by confocal laser scanning microscopy and supported by scanning electron microscopy and showed that the density of the fibers on the side adjacent to the electrode is higher, which can affect cell diffusion, while the pore size is generally the same. Bone marrow mesenchymal stem cells cultured in a 600 μm-thick scaffold formed colonies and produced conditions for cell differentiation. An in vitro study of stem cells after 7 days revealed that cell proliferation and hepatocyte growth factor release in the 600 μm-thick scaffold were higher than in the 100 μm-thick scaffold. An in vivo study of scaffolds with and without stem cells implanted subcutaneously onto the backs of recipient mice was carried out to test their biodegradation and biocompatibility over a 0–3-week period. The cells seeded onto the 600 μm-thick scaffold promoted significant neovascularization in vivo. After 3 weeks, a significant number of donor cells persisted only on the inside of the 600 μm-thick scaffold. Thus, the use of bulkier matrices allows to prolong the effect of secretion of growth factors by stem cells during implantation. These 600 μm-thick scaffolds could potentially be utilized to repair and regenerate injuries with stem cell co-culture for vascularization of implant.

Acellular Vascular Scaffolds Preloaded With Heparin and Hepatocyte Growth Factor for Small-Diameter Vascular Grafts Might Inhibit Intimal Hyperplasia

To develop small-diameter (<6 mm) scaffolds capable of accelerating rapid endothelialization and improving long-term patency rate, we created acellular vascular scaffolds preloaded with heparin and hepatocyte growth factor (HGF). Heparin was conjugated to suppress thrombogenic responses, and HGF was immobilized to induce endothelial cells (ECs) proliferation and migration. The scaffolds immobilized with heparin exhibited highly effective localization and sustained release of HGF for 30 days in vitro. We implanted this modified scaffold into the carotid artery of a rabbit model to investigate the efficacy in vivo. The acellular vascular scaffold with heparin only was used as control. After transplantation, the patency of this modified scaffold was 91.67% at 1, 3, 6, and 12 months, while the patency rate in the group with grafted heparin only was 83.33% at 1, 3, 6, and 12 months. This modified scaffold significantly stimulated ECs proliferation and the endothelium aligned in the direction of flow after 12 months. In addition, intimal hyperplasia was significantly reduced in the grafts coated with HGF compared with the control grafts. The small-diameter vascular grafts with an inner diameter of 2.5 mm preloaded with heparin and HGF may be a substitute for autologous blood vessels in clinic.

Prevascularization-free Primary Subcutaneous Transplantation of Xenogeneic Islets Coencapsulated With Hepatocyte Growth Factor.

In vitro experiments examined slow release of HGF from the chitosan gel and islet-protection effect of HGF against hypoxia. In the latter, rat islets with/without HGF (200 ng/mL) was cultured in 1% oxygen. In in vivo experiment, fabricated device with/without HGF (10 μg/device) containing rat islets was primarily transplanted to streptozotocin-induced diabetic mice subcutaneously. In vitro experiments showed sustained release of HGF for 28 d and alleviating effect of HGF on cell death and glucose-responsive insulin release after hypoxic culture. Islet + HGF mice, but not islet-alone mice, showed decreased nonfasting blood glucose and regained body weight after transplantation. In intraperitoneal glucose tolerance test, islet + HGF mice exhibited decreased fasting blood glucose (200 ± 55 mg/dL) and good blood glucose disappearance rate (K value) (0.817 ± 0.101) comparing to normal mice (123 ± 28 mg/dL and 1.074 ± 0.374, respectively). However, in islet-alone mice, fasting blood glucose was high (365 ± 172 mg/dL) and K value was indeterminable. Serum insulin in islet + HGF mice (1.58 ± 0.94 μg/L) was close to normal mice (1.66 ± 0.55 μg/L), whereas those in islet-alone mice (0.279 ± 0.076 μg/L) and diabetic mice (0.165 ± 0.079 μg/L) were low. Immunohistochemical examination showed intact insulin- and glucagon-positive islets in retrieved devices with HGF, but no intact islet was found in the device without HGF. HGF could enhance islet survival in hypoxia and enhance in vivo function of encapsulated islets after primary subcutaneous transplantation.

Hyaluronic Acid/Alginate Hydrogel Containing Hepatocyte Growth Factor and Promotion of Vocal Fold Wound Healing.

Hepatocyte growth factor (HGF) has been shown to facilitate vocal fold (VF) wound healing. This study was undertaken to determine whether the therapeutic efficacy of HGF could be enhanced by applying it in hyaluronic acid and alginate (HA/ALG) composite hydrogels into VFs after injury in a rabbit model. HGF was loaded into HA/ALG composite hydrogel (HGF-HA/ALG) and its in vitro release profile was evaluated. In addition, HGF-HA/ALG was injected into the VFs of rabbits immediately after direct injury and HGF or PBS was injected in the same manner into control groups. Macroscopic features were observed by endoscopy at 3months post-injury. Functional analyses including mucosal waves of VFs and viscoelastic properties were performed by kymography following high-speed digital imaging and rheometer. Histopathological and immunohistochemical evaluations were also conducted on VFs. HGF release from HGF-HA/ALG was sustained for up to 3weeks. Rabbits treated with HGF-HA/ALG showed improved mucosal vibrations and VF viscoelastic properties as compared with the PBS and HGF controls. Histopathological staining revealed HGF-HA/ALG treated VFs showed less fibrosis than PBS and HGF controls, and immunohistochemical analysis demonstrated amounts of type I collagen and fibronectin were lower in HGF-HA/ALG treated animals than in PBS and HGF controls at 3months post-injury. HGF containing HA/ALG hydrogel enhanced healing in our rabbit model of VF injury.

UPA alleviates kaolin-induced hydrocephalus by promoting the release and activation of hepatocyte growth factor in rats

Urokinase-type plasminogen activator (uPA) was demonstrated to alleviate kaolin-induced communicating hydrocephalus via inhibiting subarachnoid space fibrosis, but the exact mechanism remains elusive. Thus, this study was designed to investigate if hepatocyte growth factor (HGF), which plays a vital role in uPA-triggered inhibiting of fibrosis in multiple systems, is involved in this process in hydrocephalus. There were 2 parts in this study. First, hydrocephalus was induced in rats by basal cistern injection of kaolin. Then rats were treated with saline or uPA and brain tissue and CSF were collected for Western blot and enzyme-linked immuno sorbent assay (ELISA) four days later. Second, kaolin-induced hydrocephalus rats were treated with saline, uPA, uPA + PHA665752 (antagonist of HGF) or PHA665752. Some animals received MRI four weeks later and brains were used for immunofluorescence. The others were euthanized four days later for ELISA. Both levels of total and activated HGF in the CSF was increased after uPA injections, but related mRNA expression of HGF showed no statistical significance when compared with the control group. Further, the effects of uPA that alleviating ventricular enlargement, subarachnoid fibrosis and reactive astrocytosis were partially reversed by PHA665752. Moreover, PHA665752 partially abolished uPA-induced reduction of transforming growth factor- β1(TGF- β1) level in CSF. Our data suggest that uPA effectively inhibited subarachnoid fibrosis and restricted the development of communicating hydrocephalus in rats in part by promoting HGF release and activation, which may further regulate the TGF-β1 expression in CSF.

Co‐Delivery of Anti‐Inflammatory and Proliferative Agents by Injectable Hydrogel to Promote Tissue Repair after Acute Kidney Injury

ObjectIschemia‐reperfusion (I/R)‐induced acute renal injury has become a serious issue in clinical. The increasingly mortality, induced by chronic fibrosis and further renal dysfunction which caused by poor recovery of acute injury, has made it urgent to address kidney tissue repair. By using a renal I/R injury model, we aimed to investigate the role of a hydrogel‐based dual‐drug delivery platform on promoting tissue repair.MethodsAn injectable, self‐assembling peptide/heparin (SAP/Hep) hydrogel was employed to co‐deliver TNF‐α neutralizing antibody (anti‐TNF‐α) and hepatocyte growth factor (HGF). We analyzed the microstructure and controlled release properties of KLD2R/Hep hydrogel in vivo. The effects of the drug‐loaded hydrogel (SAP‐drug) on renal injury were evaluated in a rodent renal I/R injury model.ResultsIn vitro, the SAP/Hep hydrogel exhibited a faster release of anti‐TNF‐α with a sustained release of HGF, and both drugs could maintain better bioactivities after release. In vivo, combined anti‐TNF‐α/HGF showed better renal protective potential than anti‐TNF‐α or HGF alone. Compared with free‐drug (anti‐TNF‐α/HGF in solution) or SAP alone , SAP‐drug treatment significantly reduced the levels of serum creatinine (Scr)/blood urea nitrogen (BUN), tubular apoptosis, renal inflammatory factors and macrophage infiltration. Moreover, the SAP‐drug group exhibited better efficacy on promoting tubular cell proliferation and dedifferentiation than SAP or free‐drug alone, and thus reduced chronic renal fibrosis in I/R injury mice.ConclusionsWe confirmed that SAP could sequentially deliver anti‐TNF‐α and HGF to achieve anti‐inflammatory and pro‐proliferative effects simultaneously with only once injection, and thus can be a promising delivery platform for tissue repair.Support or Funding InformationThis work was supported by grants from National Natural Science Foundation of China (81571808, 31871001, 31200754) and Sichuan Science and Technology Program (2019YJ0069)Design of dual‐drug delivery KLD2R/Hep hydrogel for enhancing renal repair. The formation of the cationic KLD2R/anionic heparin hybrid hydrogel via electrostatic interaction, and heparin further had high affinity to HGF. The encapsulated anti‐TNF‐α and HGF were released from hydrogel in a sequential manner, and thus synergistically promoted tissue repair.Figure 1

Impaired integrin α5 /β1 -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5/β1 is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and β1 decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α5 and β1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin β1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5/β1 in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.

N-Cadherin Overexpression Mobilizes the Protective Effects of Mesenchymal Stromal Cells Against Ischemic Heart Injury Through a β-Catenin-Dependent Manner.

Mesenchymal stromal cell-based therapy is promising against ischemic heart failure. However, its efficacy is limited due to low cell retention and poor paracrine function. A transmembrane protein capable of enhancing cell-cell adhesion, N-cadherin garnered attention in the field of stem cell biology only recently. The current study investigates whether and how N-cadherin may regulate mesenchymal stromal cells retention and cardioprotective capability against ischemic heart failure. Adult mice-derived adipose tissue-derived mesenchymal stromal cells (ADSC) were transfected with adenovirus harboring N-cadherin, T-cadherin, or control adenovirus. CM-DiI-labeled ADSC were intramyocardially injected into the infarct border zone at 3 sites immediately after myocardial infarction (MI) or myocardial ischemia/reperfusion. ADSC retention/survival, cardiomyocyte apoptosis/proliferation, capillary density, cardiac fibrosis, and cardiac function were determined. Discovery-driven/cause-effect analysis was used to determine the molecular mechanisms. Compared with ADSC transfected with adenovirus-control, N-cadherin overexpression (but not T-cadherin) markedly increased engrafted ADSC survival/retention up to 7 days post-MI. Histological analysis revealed that ADSC transfected with adenovirus-N-cadherin significantly preserved capillary density and increased cardiomyocyte proliferation and moderately reduced cardiomyocyte apoptosis 3 days post-MI. More importantly, ADSC transfected with adenovirus-N-cadherin (but not ADSC transfected with adenovirus-T-cadherin) significantly increased left ventricular ejection fraction and reduced fibrosis in both MI and myocardial ischemia/reperfusion mice. In vitro experiments demonstrated that N-cadherin overexpression promoted ADSC-cardiomyocyte adhesion and ADSC migration, enhancing their capability to increase angiogenesis and cardiomyocyte proliferation. MMP (matrix metallopeptidases)-10/13 and HGF (hepatocyte growth factor) upregulation is responsible for N-cadherin's effect upon ADSC migration and paracrine angiogenesis. N-cadherin overexpression promotes cardiomyocyte proliferation by HGF release. Mechanistically, N-cadherin overexpression significantly increased N-cadherin/β-catenin complex formation and active β-catenin levels in the nucleus. β-catenin knockdown abolished N-cadherin overexpression-induced MMP-10, MMP-13, and HGF expression and blocked the cellular actions and cardioprotective effects of ADSC overexpressing N-cadherin. We demonstrate for the first time that N-cadherin overexpression enhances mesenchymal stromal cells-protective effects against ischemic heart failure via β-catenin-mediated MMP-10/MMP-13/HGF expression and production, promoting ADSC/cardiomyocyte adhesion and ADSC retention.

Involvement of epithelial-mesenchymal transition-inducing transcription factors in the mechanism of Helicobacter pylori-induced fibroblasts activation.

Gastric cancer (GC) originating from the lining of the stomach is one of the most frequent malignancies in humans. The most efficient method giving hope of full recovery from GC is gastric resection combined with adjuvant chemotherapy and radiotherapy. However, over 50% of patients after treatment suffer from recurrence and peritoneal metastasis. The bacteria Helicobacter pylori (Hp) is nowadays considered as the major pathogen capable of colonizing gastric mucosa. This bug causes deregulation of multiple signaling pathways including the activation of nuclear factor kappaB (NFκB) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) responsible for inflammation and development of carcinogenic cascade. The pathomechanism of these changes remains little understood, but the Hp virulence factors affecting mainly gastric epithelium have been postulated. Nevertheless, the changes associated with inflammation progressing to cancer are not only limited to epithelial cells. The cells surrounding the tumor, mainly activated fibroblasts (CAFs - cancer-associated fibroblasts) create molecular microenvironment promoting tumorigenesis and cancer invasion. The downstream targets of STAT3, epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) are expressed in activated fibroblasts providing them with additional properties. Thus, our aim was to determine if the Hp strain expressing CagA and VacA cytotoxins may result in the activation/differentiation of rat gastric fibroblasts resulting in NFκB and STAT3 signaling, which could lead to EMT-TFs expression and secretome responsible for inflammatory and EMT inducing microenvironment. In this study, gastric samples were harvested from 8-week-old Spraque-Dowley rats and the primary and secondary fibroblast cultures were established. The 70% confluent secondary fibroblast cultures were infected with 1 x 109 of live Hp expressing cytotoxins CagA VacA per dish and incubated in humidified atmosphere for 3, 24, 48 and 72 hours, before the conditioned media or the cells were used for endpoint experiments. As the control, fibroblast culture in DMEM with 10% FBS and antibiotics, free from Hp infection was used. The expression of mRNA for 18S (control), toll-like receptors: TLR2 and TLR4, STAT3, NFκB p65/Rel A, inhibitor of NF-κB (Iκβ), Snail and Twist was determined by RT-PCR. The protein expression of Snail and Twist was assessed by Western blot technique. The fibroblast supernatant was collected at 72 hours from non-infected and Hp (cagA+ vacA+)-infected culture and the concentrations of interleukin 8 (IL-8), hepatocyte growth factor (HGF) and stromal derived factor-1 (SDF-1) were measured by ELISA. In fibroblasts infected with Hp (cagA+ vacA+), the significant increase of mRNA expression for both, TLR2 and TLR4, as well as STAT3, NFκB/RelA subunit was observed already after 3 hours of fibroblasts infection with Hp strain compared with control non-infected fibroblasts. Simultaneously, the significant decrease of Iκβ mRNA has been noticed starting from 48 hours after the Hp infection of fibroblasts was carried out. The strong increase in the expression of Snail1 and Twist mRNA was recorded already at 3 hours in Hp-infected fibroblasts comparing to control non-infected fibroblasts and this increase persisted at 24 and 48 hours being the most pronounced at 72 hours post incubation with Hp. The expression of Snail1 protein was observed after 3 hours post Hp infection and this increase persisted throughout entire time periods upon Hp infection. In contrast, no detectable level of Twist protein expression was observed up to 72 hours post-infection neither in control conditions nor in fibroblasts co-infected with Hp. These changes in fibroblasts were accompanied by a significant increase in the release of HGF, SDF-1 and IL-8 determined in cell supernatants collected from Hp-infected fibroblasts. These data indicate that the activation/differentiation of rat gastric fibroblasts can occur directly by Hp releasing CagA and indirectly through TLR2 and TLR4 and these effects can be mediated by transcription factors NFκB and STAT3 signaling leading to rapid Snail1 protein expression. We conclude that NFκB and STAT3 signaling together with Snail1 protein expression may activate the secretome responsible for fibroblasts inflammatory and EMT-inducing microenvironment likely serving as prerequisite for GC development.

A self-assembling peptide hydrogel-based drug co-delivery platform to improve tissue repair after ischemia-reperfusion injury

Ischemia-reperfusion (I/R)-induced organ injury is a serious health problem worldwide, and poor recovery of acute phase injury leads to chronic fibrosis and further organ dysfunction. Thus, a more precise approach to enhance tissue repair is needed. By using a renal I/R model, we aimed to evaluate the role of a hydrogel-based dual-drug delivery platform on promoting tissue repair. An injectable, self-assembling peptide/heparin (SAP/Hep) hydrogel was used to co-deliver TNF-α neutralizing antibody (anti-TNF-α) and hepatocyte growth factor (HGF). The microstructure and controlled release properties of KLD2R/Hep hydrogel were analyzed. The effects of the drug-loaded hydrogel (SAP-drug) on renal injury were evaluated in mice with I/R injury. In vitro, the SAP/Hep hydrogel allowed for a faster release of anti-TNF-α with a sustained release of HGF, and both drugs maintained their bioactivities after release. In vivo, combined anti-TNF-α/HGF showed better renal protective potential than anti-TNF-α or HGF alone. SAP-drug (anti-TNF-α/HGF in SAP hydrogel) treatment reduced the level of serum creatinine (Scr), blood urea nitrogen (BUN), tubular apoptosis, renal inflammatory factors, and macrophage infiltration compared to Free-drug (anti-TNF-α/HGF in solution) or SAP alone. Moreover, the SAP-drug group had better efficacy on promoting tubular cell proliferation and dedifferentiation than SAP or Free-drug alone, and thus reduced chronic renal fibrosis in I/R mice. This study highlighted that SAP could sequentially deliver the two drugs to achieve anti-inflammatory and pro-proliferative effects with one injection and thus is a promising delivery platform for tissue repair. Statement of significanceIschemia-reperfusion (I/R)-induced organ injury is a serious health issue, and delayed tissue repair leads to chronic fibrosis and organ failure. Systemic administration of anti-inflammatory agents or growth factors have shown some benefits on I/R injury, but their therapeutic efficacy was limited by side effects, poor bioavailability, and absent key signals of tissue repair. To address these issues, a hydrogel-based drug co-delivery platform was used to treat I/R injury. This platform could achieve sequential release kinetics with faster rate of anti-TNF-ɑ and slower rate of HGF, and effectively promoted tissue repair by targeting inflammation and proliferation in mice with renal I/R. This nanoscale delivery platform represents a promising strategy for solid organs (heart, liver and kidney) regeneration after I/R

Co‑culturing with hypoxia pre‑conditioned mesenchymal stem cells as a new strategy for the prevention of irradiation‑induced fibroblast‑to‑myofibroblast transition.

Cardiac fibrosis is a pathological consequence of radiation-induced fibroblast proliferation and fibroblast-to-myofibroblast transition (FMT). Mesenchymal stem cell (MSC) transplantation has been revealed to be an effective treatment strategy to inhibit cardiac fibrosis. We identified a novel MSC-driven mechanism that inhibited cardiac fibrosis, via the regulation of multiple fibrogenic pathways. Hypoxia pre-conditioned MSCs (MSCsHypoxia) were co-cultured with fibroblasts using a Transwell system. Radiation-induced fibroblast proliferation was assessed using an MTT assay, and FMT was confirmed by assessing the mRNA levels of various markers of fibrosis, including type I collagen (Col1) and alpha smooth muscle actin (α-SMA). α-SMA expression was also confirmed via immunocytochemistry. The expression levels of Smad7 and Smad3 were detected by western blotting, and Smad7 was silenced using small interfering RNAs. The levels of oxidative stress following radiation were assessed by the detection of reactive oxygen species (ROS) and the activity of superoxide dismutase (SOD), malondialdehyde (MDA), and 4-hydroxynonenal (HNE). It was revealed that co-culturing with MSCsHypoxia could inhibit fibroblast proliferation and FMT. In addition, the present results indicated that MSCs are necessary and sufficient for the inhibition of fibroblast proliferation and FMT by functionally targeting TGF-β1/Smad7/Smad3 signaling via the release of hepatocyte growth factor (HGF). Furthermore, it was observed that MSCs inhibited fibrosis by modulating oxidative stress. Co-culturing with MSCsHypoxia alleviated fibroblast proliferation and FMT via the TGF-β1/Smad7/Smad3 pathway. MSCs may represent a novel therapeutic approach for the treatment of radiation-related cardiac fibrosis.

Our Steps toward Subcutaneous Transplantation of Macro-Encapsulated Islets

Background: Diabetes mellitus (DM) can be cured or greatly ameliorated by adequate insulin secretion from a relatively small volume of insulin-producing cells. Cell encapsulation enables allo- and even xeno-geneic cell therapy without immunosuppression. However, micro-encapsulated islets used in recent clinical trials are not fully retrievable after transplantation. By contrast, macro-encapsulated islets can be retrievable. As to the transplantation site, the subcutaneous tissue can be promising, if new strategy could overcome the hypoxic status due to hypovascularity. In this review article, we summarized the development of macro-encapsulated islets and approaches toward subcutaneous transplantation in our laboratory for more than two decades. Our results repeatedly showed that islets and encapsulated islets can be transplanted in various sites including subcutaneous tissue after pretreatment of neovascular induction. As to macro-encapsulation devices, our laboratory firstly developed mesh-reinforced poly-vinyl alcohol (PVA) hydrogel tube and bag and then, agarose-based encapsulation methods followed. After that, PVA macro-encapsulated islets were developed utilizing sol-gel transition of PVA solution through micro-crystallization induced by freezing and thawing. Recently, we took advantage of excellent histocompatibility of ethylene vinyl alcohol co-polymer (EVOH) to fabricate the highly porous EVOH bag. A unique macro-encapsulation device using thermo-sensitive chitosan-based gel in combination with EVOH bag was developed. Most recently, we found that slow release of hepatocyte growth factor (HGF) from the chitosan gel can protect islets against initial hypoxic status and subsequently induce neovascularization in subcutaneous tissue to exert islet function, eliminating the necessity of neovascularization pretreatment. Conclusions: Retrievable and replaceable macro-encapsulated islets that can be transplanted subcutaneously without pretreatment. Although further improvements may be necessary, this method may replace intraportal islet transplantation with immunosuppression in the near future.

Coronary stents with inducible VEGF/HGF-secreting UCB-MSCs reduced restenosis and increased re-endothelialization in a swine model

Atherosclerotic plaques within the vasculature may eventually lead to heart failure. Currently, cardiac stenting is the most effective and least invasive approach to treat this disease. However, in-stent restenosis is a complex chronic side effect of stenting treatment. This study used coronary stents coated with stem cells secreting angiogenic growth factors via an inducible genome-editing system to reduce stent restenosis and induce re-endothelialization within the artery. The characteristics of the cells and their adhesion properties on the stents were confirmed, and the stents were transplanted into a swine model to evaluate restenosis and the potential therapeutic use of stents with stem cells. Restenosis was evaluated using optical coherence tomography (OCT), microcomputed tomography (mCT) and angiography, and re-endothelialization was evaluated by immunostaining after cardiac stent treatment. Compared to a bare metal stent (BMS) or a parental umbilical cord blood-derived mesenchymal stem cell (UCB-MSC)-coated stent, the stents with stem cells capable of the controlled release of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) successfully reduced restenosis within the stent and induced natural re-endothelialization. Furthermore, UCB-MSCs exhibited the ability to differentiate into endothelial cells in Matrigel, and HGF and VEGF improved this differentiation. Our study indicates that stents coated with UCB-MSCs secreting VEGF/HGF reduce the restenosis side effects of cardiac stenting with improved re-endothelialization.