Key inflammatory markers in bronchoalveolar lavage predict bronchiectasis progression in young children with CF.

INTRODUCTION: Inflammation appears early in cystic fibrosis (CF) pathogenesis, with specific elevated inflammatory markers in bronchoalveolar lavage fluid (BALF) correlating with structural lung disease. Our aim was to identify markers of airway inflammation able to predict bronchiectasis progression over two years with high sensitivity and specificity. METHODS: Children with CF with two chest computed tomography (CT) scans and bronchoscopies at a two-year interval were included (n= 10 at 1 and 3 years and n= 27 at 3 and 5 years). Chest CTs were scored for increase in bronchiectasis (Δ%Bx), using the PRAGMA-CF score. BALF collected with the first CT scan were analyzed for neutrophil% (n= 36), myeloperoxidase (MPO) (n= 25), neutrophil elastase (NE) (n= 26), and with a protein array for inflammatory and fibrotic markers (n= 26). RESULTS: MPO, neutrophil%, and inducible T-cell costimulator ligand (ICOSLG), but not clinical characteristics, correlated significantly with Δ%Bx. Evaluation of neutrophil%, NE, MPO, interleukin-8 (IL-8), ICOSLG, and hepatocyte growth factor (HGF), for predicting an increase of > 0.5% of Δ%Bx in two years, showed that IL-8 had the best sensitivity (82%) and specificity (73%). Neutrophil%, ICOSLG and HGF had sensitivities of 85, 82, and 82% and specificities of 59, 67 and 60%, respectively. The odds ratio for risk of >0.5% Δ%Bx was higher for IL-8 (12.4) than for neutrophil%, ICOSLG, and HGF (5.9, 5.3, and 6.7, respectively). Sensitivity and specificity were lower for NE and MPO). CONCLUSIONS: High levels of IL-8, neutrophil%, ICOSGL and HGF in BALF may be good predictors for progression of bronchiectasis in young children with CF.

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.

EXPRESSION OF HEPATOCYTE GROWTH FACTOR AND C-MET RECEPTOR IN STROMAL FIBROBLASTS AND TUMOR CELLS OF ENDOMETRIAL CARCINOMA.

BACKGROUND: HGF/c-Met is one of the main signaling pathways that ensure communication between epithelial cells and components of the tumor microenvironment determining the invasive and metastatic potential of many cancers. However, the significance of HGF and c-Met in endometrial carcinoma (ECa) progression remains unclear. AIM: To evaluate copy number variations as well as expression of the c-Met receptor and its ligand HGF in endometrial carcinomas considering the clinical and morphological characteristics of ECa. MATERIALS AND METHODS: The study was conducted on ECa samples of 57 patients, among which 32 had lymph nodes and/or distant metastasis. The copy number of c-MET gene was estimated by qPCR. The expression of HGF and c-Met in tissue samples was determined by the immunohistochemical method. RESULTS: Amplification of c-MET gene was detected in 10.5% of the ECa cases. In most carcinomas, a combined expression pattern of HGF and c-Met was established, in which co-expression of these markers was observed in tumor cells, and the content of HGF+ fibroblasts increased in the stroma. The expression of HGF in tumor cells was associated with the tumor differentiation grade and was higher in G3 ECa (p = 0.041). The number of HGF+ fibroblasts in the stromal component increased in the ECa cases with metastasis compared to the cases without metastasis (p = 0.032). The content of stromal c-Met+ fibroblasts was higher in deeply invasive carcinomas of patients with metastases than in tumors with invasion of < 1/2 myometrium (p = 0.035). CONCLUSION: Increased expression of HGF and c-Met in stromal fibroblasts of endometrial carcinomas is associated with metastasis in patients with ECa and deep invasion of the tumor into the myometrium, and can contribute to the aggressive course of the disease.

Immunofibrotic drivers of impaired lung function in postacute sequelae of SARS-CoV-2 infection.

BACKGROUNDIndividuals recovering from COVID-19 frequently experience persistent respiratory ailments, which are key elements of postacute sequelae of SARS-CoV-2 infection (PASC); however, little is known about the underlying biological factors that may direct lung recovery and the extent to which these are affected by COVID-19 severity.METHODSWe performed a prospective cohort study of individuals with persistent symptoms after acute COVID-19, collecting clinical data, pulmonary function tests, and plasma samples used for multiplex profiling of inflammatory, metabolic, angiogenic, and fibrotic factors.RESULTSSixty-one participants were enrolled across 2 academic medical centers at a median of 9 weeks (interquartile range, 6-10 weeks) after COVID-19 illness: n = 13 participants (21%) had mild COVID-19 and were not hospitalized, n = 30 participants (49%) were hospitalized but were considered noncritical, and n = 18 participants (30%) were hospitalized and in the intensive care unit (ICU). Fifty-three participants (85%) had lingering symptoms, most commonly dyspnea (69%) and cough (58%). Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and diffusing capacity for carbon monoxide (DLCO) declined as COVID-19 severity increased (P < 0.05) but these values did not correlate with respiratory symptoms. Partial least-squares discriminant analysis of plasma biomarker profiles clustered participants by past COVID-19 severity. Lipocalin-2 (LCN2), MMP-7, and HGF identified by our analysis were significantly higher in the ICU group (P < 0.05), inversely correlated with FVC and DLCO (P < 0.05), and were confirmed in a separate validation cohort (n = 53).CONCLUSIONSubjective respiratory symptoms are common after acute COVID-19 illness but do not correlate with COVID-19 severity or pulmonary function. Host response profiles reflecting neutrophil activation (LCN2), fibrosis signaling (MMP-7), and alveolar repair (HGF) track with lung impairment and may be novel therapeutic or prognostic targets.FundingNational Heart, Lung, and Blood Institute (K08HL130557 and R01HL142818), American Heart Association (Transformational Project Award), the DeLuca Foundation Award, a donation from Jack Levin to the Benign Hematology Program at Yale University, and Duke University.

Hepatocyte Growth Factor and Incident Heart Failure Subtypes: The Multi-Ethnic Study of Atherosclerosis (MESA).

BACKGROUND: Hepatocyte growth factor (HGF) is a cytokine and marker of cardiovascular disease (CVD) risk. Less is known about HGF and incident heart failure (HF). We examined the association of HGF with incident HF and its subtypes in a multiethnic cohort. METHODS AND RESULTS: We included 6597 participants of the Multi-Ethnic Study of Atherosclerosis (MESA) cohort, free of clinical CVD and HF at baseline, with HGF measured at baseline. Incident hospitalized HF was assessed and adjudicated for HF with preserved ejection fracture (HFpEF) vs HF with reduced ejection fraction (HFrEF). Cox regression models estimated hazard ratios (HR) and 95% confidence intervals (CI) for HF risk by HGF levels, adjusted for socio-demographics, CVD risk factors and N-terminal pro-B-type natriuretic peptide. The mean age was 62 ± 10 years. The median HGF level was 950 pg/mL (interquartile range, 758-1086 pg/mL); 53% were women. Over 14 years (IQR, 11.5-14.7 years), there were 324 cases of HF (133 HFpEF and 157 HFrEF). For the highest HGF tertile compared with lowest, adjusted HRs were 1.59 (95% CI, 1.10-2.31), 1.90 (95% CI, 1.03-3.51), and 1.09 (95% CI, 0.65-1.82) for overall HF, HFpEF, and HFrEF, respectively. For continuous analysis per 1-standard deviation log-transformed HGF, adjusted HRs were 1.22 (95% CI, 1.06-1.41), 1.35 (95% CI, 1.09-1.69), and 1.00 (95% CI, 0.81-1.24) for HF, HFpEF, and HFrEF, respectively. CONCLUSIONS: HGF was independently associated with incident HF. HGF remained significantly associated with HFpEF but not HFrEF upon subtype assessment. Future studies should examine the mechanisms underlying these associations and evaluate whether HGF can be used to improve HF risk prediction or direct therapy.

Effect of prolactin on normal and keratoconus human corneal stromal fibroblasts in vitro.

PURPOSE: To examine the effect of prolactin (PRL) on human corneal stromal fibroblasts (CSFs), derived from healthy individuals and from keratoconus (KC) patients, in vitro, specifically assessing physiological and elevated PRL concentrations as apparent during pregnancy. METHODS: Eye bank corneas of 3 female and 3 male healthy individuals as well as the corneal buttons of 3 female and 3 male KC patients were utilized for this study. The endothelium of the cornea was removed with sterile surgical scalpels, the probes were washed repeatedly with Dulbecco's PBS and corneoscleral rims were trimmed off. Subsequently the corneal stroma was digested with collagenase type I and the harvested CSFs were cultured. We then examined (1) cell proliferation, (2) cell viability and (3) cytokine release of CSFs upon exposure to prolactin in vitro. RESULTS: With respect to viability and proliferation our experiments did not show significant differences between CSFs exposed to different PRL concentrations. Our data show a significantly lower IL-8 concentration in normal CSFs exposed to 10ng/ml PRL compared to 0ng/ml and 1000ng/ml at 5 hours post exposition. Moreover, we can report significantly lower secretion of IL-8, IL-6, HGF, VEGF and FGFb in KC CSFs compared to normal CSFs, independent of PRL exposure, as determined by cytokine ELISA. CONCLUSION: Our data in part points towards corneal cytokine secretion as a possible link between altered stromal PRL concentrations and KC progression. However, in our small dataset a significant influence of PRL concentration on cytokine secretion can only be described for IL-8 in normal CSFs. Further our results contribute to existing reports on the importance of cytokines in KC development, with an emphasis on significantly lower cytokine secretion in KC CSFs compared to normal controls.

Localized Cell-Surface Sampling of a Secreted Factor Using Cell-Targeting Beads.

Intercellular communication through the secretion of soluble factors plays a vital role in a wide range of biological processes (e.g., homeostasis, immune response), yet identification and quantification of many of these factors can be challenging due to their degradation or sequestration in cell culture media prior to analysis. Here, we present a customizable bead-based system capable of simultaneously binding to live cells (through antibody-mediated cell tethering) and capturing cell-secreted molecules. Our functionalized beads capture secreted molecules (e.g., hepatocyte growth factor secreted by fibroblasts) that are diminished when sampled via traditional supernatant analysis techniques (p < 0.05), effectively rescuing a reduced signal in the presence of neutralizing components in the cell culture media. Our system enables capture and analysis of molecules integral to chemical communication that would otherwise be markedly decreased prior to analysis.

Expression of HGF, pMet, and pAkt is related to benefit of radiotherapy after breast-conserving surgery: a long-term follow-up of the SweBCG91-RT randomised trial.

Experimental studies suggest that hepatocyte growth factor (HGF) and its transmembrane tyrosine kinase receptor, Met, in part also relying on Akt kinase activity, mediate radioresistance. We investigated the importance of these biomarkers for the risk of ipsilateral breast tumour recurrence (IBTR) after adjuvant radiotherapy (RT) in primary breast cancer. HGF, phosphorylated Met (pMet) and phosphorylated Akt (pAkt) were evaluated immunohistochemically on tissue microarrays from 1004 patients in the SweBCG91-RT trial, which randomly assigned patients to breast-conserving therapy, with or without adjuvant RT. HGF was evaluated in the stroma (HGFstr ); pMet in the membrane (pMetmem ); HGF, pMet and pAkt in the cytoplasm (HGFcyt , pMetcyt , pAktcyt ); and pAkt in the nucleus (pAktnuc ). The prognostic and treatment predictive effects were evaluated to primary endpoint IBTR as first event during the first 5 years. Patients with tumours expressing low levels of HGFcyt and pMetcyt and high levels of pAktnuc derived a larger benefit from RT [hazard ratio (HR): 0.11 (0.037-0.30), 0.066 (0.016-0.28) and 0.094 (0.028-0.31), respectively] compared to patients with high expression of HGFcyt and pMetcyt , and low pAktnuc [HR: 0.36 (0.19-0.67), 0.35 (0.20-0.64) and 0.47 (0.32-0.71), respectively; interaction analyses: P = 0.052, 0.035 and 0.013, respectively]. These differences remained in multivariable analysis when adjusting for patient age, tumour size, histological grade, St Gallen subtype and systemic treatment (interaction analysis, P-values: 0.085, 0.027, and 0.023, respectively). This study suggests that patients with immunohistochemically low HGFcyt , low pMetcyt and high pAktnuc may derive an increased benefit from RT after breast-conserving surgery concerning the risk of developing IBTR.

Longitudinal Changes of NF-κB Downstream Mediators and Peritoneal Transport Characteristics in Incident Peritoneal Dialysis Patients.

The role of intra-peritoneal mediators in the regulation peritoneal transport is not completely understood. We investigate the relation between longitudinal changes in dialysis effluent level of nuclear factor kappa-B (NF-κB) downstream mediators and the change in peritoneal transport over 1 year. We studied 46 incident PD patients. Their peritoneal transport characteristics were determined after starting PD and then one year later. Concomitant dialysis effluent levels of interleukin-6 (IL-6), cyclo-oxygenase-2 (COX-2) and hepatocyte growth factor (HGF) are determined. There were significant correlations between baseline and one-year dialysis effluent IL-6 and COX-2 levels with the corresponding dialysate-to-plasma creatinine level at 4 hours (D/P4) and mass transfer area coefficient of creatinine (MTAC). After one year, patients who had peritonitis had higher dialysis effluent IL-6 (26.6 ± 17.4 vs 15.1 ± 12.3 pg/ml, p = 0.037) and COX-2 levels (4.97 ± 6.25 vs 1.60 ± 1.53 ng/ml, p = 0.007) than those without peritonitis, and the number of peritonitis episode significantly correlated with the IL-6 and COX-2 levels after one year. In contrast, dialysis effluent HGF level did not correlate with peritoneal transport. There was no difference in any mediator level between patients receiving conventional and low glucose degradation product solutions. Dialysis effluent IL-6 and COX-2 levels correlate with the concomitant D/P4 and MTAC of creatinine. IL-6 and COX-2 may contribute to the short-term regulation of peritoneal transport.

Transplantation of human adipose tissue derived-SVF enhance liver function through high anti-inflammatory property.

Although human adipose tissue-derived stromal vascular fraction (SVF) has been considered a promising source of stem cells, its characteristics relevant to treatment of a damaged liver have not been fully elucidated. In the present study, we sought to characterize the property of human SVF and determine the therapeutic utility of SVF in the liver cirrhosis model. We performed microarray, quantitative (q)-PCR experiments, and in vivo therapeutic assays using a liver cirrhotic mouse model. q-PCR results revealed that hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)-A, Interleukin (IL)-10 and microRNA (miR)-146 were more highly upregulated in SVF than in human adipose-derived mesenchymal stem cells (ASCs). The SVF culture medium (CM) inhibited the activation of hepatic stellate cells in vitro. Injection of SVF significantly suppressed TAA-induced liver fibrosis and repaired liver function by inhibition of infiltrating inflammatory cells and induction of capillary/hepatocyte regeneration in vivo. Injection of IL-10 siRNA treated SVF cells decreased anti-inflammation and anti-fibrotic effects in TAA-induced mice liver. Our data indicate that SVF show a high anti-inflammatory property for treating fibrotic liver diseases through IL-10 secretion. Therefore, SVF might be a novel therapeutic alternative for the treatment of liver cirrhosis in clinical settings.

Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor.

Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF-MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.

Lung fluid biomarkers for acute respiratory distress syndrome: a systematic review and meta-analysis.

BACKGROUND: With the development of new techniques to easily obtain lower respiratory tract specimens, bronchoalveolar lavage fluid and other lung fluids are gaining importance in pulmonary disease diagnosis. We aimed to review and summarize lung fluid biomarkers associated with acute respiratory distress syndrome diagnosis and mortality. METHODS: After searching PubMed, Embase, Web of Science, and the Cochrane Library for articles published prior to January 11, 2018, we performed a meta-analysis on biomarkers for acute respiratory distress syndrome diagnosis in at-risk patients and those related to disease mortality. From the included studies, we then extracted the mean and standard deviation of the biomarker concentrations measured in the lung fluid, acute respiratory distress syndrome etiologies, sample size, demographic variables, diagnostic criteria, mortality, and protocol for obtaining the lung fluid. The effect size was measured by the ratio of means, which was then synthesized by the inverse-variance method using its natural logarithm form and transformed to obtain a pooled ratio and 95% confidence interval. RESULTS: In total, 1156 articles were identified, and 49 studies were included. Increases in total phospholipases A2 activity, total protein, albumin, plasminogen activator inhibitor-1, soluble receptor for advanced glycation end products, and platelet activating factor-acetyl choline were most strongly associated with acute respiratory distress syndrome diagnosis. As for biomarkers associated with acute respiratory distress syndrome mortality, interleukin-1ß, interleukin-6, interleukin-8, Kerbs von Lungren-6, and plasminogen activator inhibitor-1 were significantly increased in the lung fluid of patients who died. Decreased levels of Club cell protein and matrix metalloproteinases-9 were associated with increased odds for acute respiratory distress syndrome diagnosis, whereas decreased levels of Club cell protein and interleukin-2 were associated with increased odds for acute respiratory distress syndrome mortality. CONCLUSIONS: This meta-analysis provides a ranking system for lung fluid biomarkers, according to their association with diagnosis or mortality of acute respiratory distress syndrome. The performance of biomarkers among studies shown in this article may help to improve acute respiratory distress syndrome diagnosis and outcome prediction.

Substance P Promotes Liver Sinusoidal Endothelium-Mediated Hepatic Regeneration by NO/HGF Regulation.

Liver sinusoidal endothelial cells (LSECs) are highly specialized and involved in hepatic regeneration by interacting with hepatic stellate cells (HSCs) and hepatocytes in a paracrine manner. However, hepatic injury can impair cellular activity and lead to endothelial dysfunction, eventually inducing the development of critical hepatic disease, including cirrhosis. Because LSECs exert their effects through paracrine factors, maintenance of paracrine potentials and survival activity in LSECs under injury stress is a critical strategy for inhibiting disease progression. This study explored the effect of Substance-P (SP) on cell viability, proliferation, and nitric oxide (NO)/hepatocyte growth factor (HGF) production in LSECs. Under noninjured conditions, SP treatment enhanced cell viability, cell proliferation, and HGF/NO secretion in LSECs. In the presence of tumor necrosis factor (TNF)-α-induced inflammatory stress, SP blocked TNF-α-induced endothelial dysfunction, accompanied by elevated cell viability and NO/HGF secretion. Interestingly, SP-primed LSEC-conditioned medium accelerated hepatocyte repopulation without causing morphological alterations. The primitive effect of SP was reversed by endothelial nitric oxide synthase inhibitor or HGF/c-MET inhibitor, indicating the importance of the NO/HGF combination in hepatic regeneration by SP. Taken together, these results suggest SP can protect LSECs from inflammatory stress by NO/HGF, which contributes to hepatocyte repopulation.

Clinicopathologic Features and Prognostic Factors in Alpha-Fetoprotein-Producing Colorectal Cancer: Analysis of 78 Cases.

BACKGROUND/AIMS: Alpha-fetoprotein-producing colorectal cancer (AFPP-CRC) is quite rarely seen. This study aimed to elucidate the clinicopathologic characteristics and prognostic factors of AFPP-CRC. METHODS: Among 5,051 colorectal cancer patients receiving surgery in the Fudan University Shanghai Cancer Center from 2006 to 2016, we identified 78 patients with elevated serum level of AFP (> 10 µg/L) preoperatively. A propensity score matching (PSM) analysis was performed which matched 75 AFPP-CRC patients to the same number of AFP-negative colorectal cancer (AFPN-CRC) patients. Kaplan-Meier curves were compared using the log-rank test and multivariable analysis was performed to evaluate the effect of AFP-positivity while adjusting confounding factors. 27 patients were available for immunohistochemical analysis. We conducted functional experiments to characterize the tumorigenicity of AFP. RESULTS: Patients with AFPP-CRC had a significantly higher incidence of advanced TNM stage and liver metastasis. Overall survival was significantly different between two groups before and after PSM, and AFP-positivity was one of the strongest predictors of overall survival in the multivariable model (HR 4.11, CI 95%: 1.43-11.76, p = 0.009) after PSM. We further investigated prognostic factors affecting prognosis in AFPP-CRC and found that the presence of liver metastasis was the only independent prognostic factor (HR 4.95, CI 95%: 1.48-16.48, p = 0.009). AFP expression was significantly positively correlated with HGF and c-Met expression. Transwell invasion assay revealed significantly increased cell motility with AFP overexpression. CONCLUSION: AFP-positivity is a significant negative predictor of overall survival in patients with colorectal cancer, which may be mediated by HGF/c-Met signaling pathway.

A pilot study to profile salivary angiogenic factors to detect head and neck cancers.

BACKGROUND: Early diagnosis of head and neck squamous cell carcinoma (HNSCCs) is an appealing way to increase survival rates in these patients as well as to improve quality of life post-surgery. Angiogenesis is a hallmark of tumor initiation and progression. We have investigated a panel of angiogenic factors in saliva samples collected from HNSCC patients and controls using the Bio-Plex ProTM assays. METHODS: We have identified a panel of five angiogenic proteins (sEGFR, HGF, sHER2, sIL-6Ra and PECAM-1) to be elevated in the saliva samples collected from HNSCC patients (n = 58) compared to a control cohort (n = 8 smokers and n = 30 non-smokers). RESULTS: High positive correlations were observed between the following sets of salivary proteins; sEGFR:sHER2, sEGFR:HGF, sEGFR:sIL-6Rα, sHER2:HGF and sHER2:sIL6Ra. A moderate positive correlation was seen between FGF-basic and sEGFR. CONCLUSION: We have shown that angiogenic factor levels in saliva can be used as a potential diagnostic biomarker panel in HNSCC.

Partial Inhibition of HO-1 Attenuates HMP-Induced Hepatic Regeneration against Liver Injury in Rats.

We found better liver graft regeneration with hypothermic machine perfusion (HMP) compared with static cold storage (SCS) for the first time in our pilot study, but the underlying mechanisms are unknown. Upregulated heme oxygenase- (HO-) 1 expression has been reported to play a pivotal role in promoting hepatocyte proliferation. Here, we evaluated the novel role of HO-1 in liver graft protection by HMP. Rats with a heterozygous knockout of HO-1 (HO-1+/-) were generated and subjected to 3 h of SCS or HMP pre-half-size liver transplantation (HSLT) in vivo or 6 h of SCS or HMP in vitro; control rats were subjected to the same conditions (HO-1+/+). We found that HSLT induced significant elevation of the HO-1 protein level in the regenerated liver and that HO-1 haplodeficiency resulted in decreased proliferation post-HSLT. Compared with SCS, HMP induced significant elevation of the HO-1 protein level along with better liver recovery, both of which were reduced by HO-1 haplodeficiency. HO-1 haplodeficiency-induced decreased proliferation was responsible for the attenuated regenerative ability of HMP. Mechanistically, HO-1 haploinsufficiency resulted in suppression of hepatocyte growth factor (HGF)/Akt activity. Our results suggest that inhibition of HO-1 mitigates HMP-induced liver recovery effects related to proliferation, in part, by downregulating the HGF-Akt axis.

Decellularized Diaphragmatic Muscle Drives a Constructive Angiogenic Response In Vivo.

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31⁺, αSMA⁺, and vWF⁺ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF⁺ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration.

Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs.

OBJECTIVES: Decellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity. DESIGN: Human periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/- DNase besides Freeze-thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays. RESULTS: DNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention. CONCLUSIONS: This study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention.

Predictive value for lymph node metastasis of epithelial-mesenchymal transition and cancer stem cell marker expression in early gastric cancer.

BACKGROUND: Minimally invasive therapies, including endoscopic mucosal resection or sentinel node navigation surgery, have been widely applied in early gastric cancer because of their benefits in promoting patient quality of life. However, lymph node dissection is beyond the capability of endoscopic therapy, and in sentinel node navigation surgery, the potential for skip metastasis is not negligible. Therefore, the possibility of lymph node metastasis is the most important factor to consider when deciding whether to apply the minimally invasive therapies. In the present study, the significance of epithelial mesenchymal transition and stem cell marker expression in lymph node metastasis in early gastric cancer was investigated. METHODS: We evaluated the significance of the expression of 5 epithelial mesenchymal transition-related markers (E-cadherin, MMP7, S100A, Snail-1, and HGF) and 6 stem cell markers (ALDH1, SOX2, CD24, CD44, CD54, and CD133) in 119 early gastric cancer specimens using immunohistochemistry. Because protein expression is heterogeneous in gastric cancer, we analyzed the expression of these markers in two selected regions (one each at the superficial zone and the deep invasive front). RESULTS: Expression of E-cadherin, MMP7, HGF, and CD133 at the deep invasive front was associated with the absence of lymph node metastasis (P=0.013, 0.018, <0.001, and 0.026, respectively). Presence of diffuse-type component, lymphatic invasion, and lack of expression of HGF and CD133 at the deep invasive front were independent predictive markers of lymph node metastasis (P=0.019, <0.001, 0.015, and 0.047, respectively). CONCLUSIONS: Lymph node metastasis is strongly associated with expression status of HGF and CD133 at the deep invasive front, suggesting the usefulness of these proteins as independent predictive markers of lymph node metastasis in early gastric cancer.

Immune Components in Human Milk Are Associated with Early Infant Immunological Health Outcomes: A Prospective Three-Country Analysis.

The role of breastfeeding in improving allergy outcomes in early childhood is still unclear. Evidence suggests that immune mediators in human milk (HM) play a critical role in infant immune maturation as well as protection against atopy/allergy development. We investigated relationships between levels of immune mediators in colostrum and mature milk and infant outcomes in the first year of life. In a large prospective study of 398 pregnant/lactating women in the United Kingdom, Russia and Italy, colostrum and mature human milk (HM) samples were analysed for immune active molecules. Statistical analyses used models adjusting for the site of collection, colostrum collection time, parity and maternal atopic status. Preliminary univariate analysis showed detectable interleukin (IL) 2 and IL13 in HM to be associated with less eczema. This finding was further confirmed in multivariate analysis, with detectable HM IL13 showing protective effect OR 0.18 (95% CI 0.04-0.92). In contrast, a higher risk of eczema was associated with higher HM concentrations of transforming growth factor ß (TGFß) 2 OR 1.04 (95% CI 1.01-1.06) per ng/mL. Parental-reported food allergy was reported less often when IL13 was detectable in colostrum OR 0.10 (95% CI 0.01-0.83). HM hepatocyte growth factor (HGF) was protective for common cold incidence at 12 months OR 0.19 (95% CI 0.04-0.92) per ng/mL. Data from this study suggests that differences in the individual immune composition of HM may have an influence on early life infant health outcomes. Increased TGFß2 levels in HM are associated with a higher incidence of reported eczema, with detectable IL13 in colostrum showing protective effects for food allergy and sensitization. HGF shows some protective effect on common cold incidence at one year of age. Future studies should be focused on maternal genotype, human milk microbiome and diet influence on human milk immune composition and both short- and long-term health outcomes in the infant.