Release Of Fibronectin Research Articles

Fibronectin as a predictor of preeclampsia

Since preeclampsia first emerged a few centuries ago, a substantial portion of humanity has been tormented by this disease, which has jeopardized and disrupted mankind. Numerous biological markers, including ENDOGLIN, PIGF (placental growth factor), SFLT 1(soluble fms like-tyrosine kinase 1), and PAPP-A (pregnancy associated plasma protein-A), suggest preeclampsia. It was beneficial for detecting preeclampsia, even if there was frequent mortality and morbidity among mothers because of the anticipated latency. Then another marker, fibronectin, was added to the picture. A better preeclampsia prognosis leads substantially to an overall decrease in maternal morbidity and mortality. The primary organ that manufactures circulating fibronectin, occasionally referred to as serum fibronectin, is the liver. To enable the extracellular and intracellular matrix to communicate and regulate cell behavior, fibronectin, an extracellular matrix element, primarily interacts with integrin receptors. Fibronectin is a diagnostic marker for a variety of ailments, such as muscular dystrophy, ovarian and stomach cancers, and gestational diabetes. Inflammatory changes result in the release of fibronectin into the bloodstream, and the main triggering factor of preeclampsia is the infiltration of various trophoblasts that injures endothelial tissues and induces inflammation. Therefore, the primary goal of our study was to establish that fibronectin is a biomarker of preeclampsia. To elucidate our perspective and evaluate whether fibronectin is relevant in preeclampsia prediction, we will conduct a qualitative evaluation of prior research and juxtapose it with the findings of the present study. Thus, early detection of preeclampsia could decrease the rate of maternal mortality and morbidity.

TMAO enhances TNF-α mediated fibrosis and release of inflammatory mediators from renal fibroblasts

Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived metabolite and TNF-α is proinflammatory cytokine, both known to be associated with renal inflammation, fibrosis and chronic kidney disease. However, today there are no data showing the combined effect of TMAO and TNF-α on renal fibrosis-and inflammation. The aim of this study was to investigate whether TMAO can enhance the inflammatory and fibrotic effects of TNF-α on renal fibroblasts. We found that the combination of TNF-α and TMAO synergistically increased fibronectin release and total collagen production from renal fibroblasts. The combination of TMAO and TNF-α also promoted increased cell proliferation. Both renal proliferation and collagen production were mediated through Akt/mTOR/ERK signaling. We also found that TMAO enhanced TNF-α mediated renal inflammation by inducing the release of several cytokines (IL-6, LAP TGF-beta-1), chemokines (CXCL-6, MCP-3), inflammatory-and growth mediators (VEGFA, CD40, HGF) from renal fibroblasts. In conclusion, we showed that TMAO can enhance TNF-α mediated renal fibrosis and release of inflammatory mediators from renal fibroblasts in vitro. Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.

Chuanxiong Rhizoma extracts prevent cholestatic liver injury by targeting H3K9ac-mediated and cholangiocyte-derived secretory protein PAI-1 and FN.

Chuanxiong Rhizoma (CX, the dried rhizome of Ligusticum wallichii Franch.), a well-known traditional Chinese medicine, is clinically used for treating cardiovascular, cerebrovascular and hepatobiliary diseases. Cholestatic liver damage is one of the chronic liver diseases with limited effective therapeutic strategies. Currently, little is known about the mechanism links between CX-induced anti-cholestatic action and intercellular communication between cholangiocytes and hepatic stellate cells (HSCs). The study aimed to evaluate the hepatoprotective activity of different CX extracts including the aqueous, alkaloid, phenolic acid and phthalide extracts of CX (CXAE, CXAL, CXPA and CXPHL) and investigate the intercellular communication-related mechanisms by which the most effective extracts work on cholestatic liver injury. The active compounds of different CX extracts were identified by UPLC-MS/MS. A cholestatic liver injury mouse model induced by bile duct ligation (BDL), and transforming growth factor-β (TGF-β)-treated human intrahepatic biliary epithelial cholangiocytes (HIBECs) and HSC cell line (LX-2 cells) were used for in vivo and in vitro studies. Histological and other biological techniques were also applied. The results indicated that CXAE, CXAL and CXPHL significantly reduced ductular reaction (DR) and improved liver fibrosis in the BDL mice. Meanwhile, both CXAE and CXPHL suppressed DR in injured HIBECs and reduced collagen contraction force and the expression of fibrosis biomarkers in LX-2 cells treated with TGF-β. CXPHL suppressed the transcription and transfer of plasminogen activator inhibitor-1 (PAI-1) and fibronectin (FN) from the 'DR-like' cholangiocytes to activated HSCs. Mechanistically, the inhibition of PAI-1 and FN by CXPHL was attributed to the untight combination of the acetyltransferase KAT2A and SMAD3, followdd by the suppression of histone 3 lysine 9 acetylation (H3K9ac)-mediated transcription in cholangiocytes. In conclusion, CXPHL exerts stronger anti-cholestatic activity in vivo and in vitro than other CX extracts, and its protective effect on the intracellular communication between cholangiocytes and HSCs is achieved by reducing KAT2A/H3K9ac-mediated transcription and release of PAI-1 and FN.

The Fibrotic Effects of TMAO on Human Renal Fibroblasts Is Mediated by NLRP3, Caspase-1 and the PERK/Akt/mTOR Pathway.

Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, has previously been shown to be implicated in chronic kidney disease. A high TMAO-containing diet has been found to cause tubulointerstitial renal fibrosis in mice. However, today there are no data linking specific molecular pathways with the effect of TMAO on human renal fibrosis. The aim of this study was to investigate the fibrotic effects of TMAO on renal fibroblasts and to elucidate the molecular pathways involved. We found that TMAO promoted renal fibroblast activation and fibroblast proliferation via the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 signaling. We also found that TMAO increased the total collagen production from renal fibroblasts via the PERK/Akt/mTOR pathway. However, TMAO did not induce fibronectin or TGF-β1 release from renal fibroblasts. We have unraveled that the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 mediates TMAO’s fibrotic effect on human renal fibroblasts. Our results can pave the way for future research to further clarify the molecular mechanism behind TMAO’s effects and to identify novel therapeutic targets in the context of chronic kidney disease.

3D Transglutaminase Fibronectin Hydrogel Therapy for Healing of Chronic Irradiated Porcine Skin Wounds

PURPOSE/BACKGROUND: Chronic irradiated wounds are characterized by incomplete healing, with no effective therapies reported for the treatment of cutaneous radiation injury. Our group previously demonstrated that fibronectin, a key extracellular matrix glycoprotein involved wound healing, is significantly downregulated in radiation-damaged skin. We further found that an enzymatically crosslinked hydrogel is a suitable construct for sustained and incremental fibronectin release. Our present objective was to investigate the application of this fibronectin hydrogel dressing for the treatment of irradiated wounds in the clinically relevant porcine irradiated wound model. METHODS: We created a chronic irradiation skin injury model in female Yucatan minipigs. Six 1-month-old minipigs underwent irradiation of the right dorsolateral neck region for five consecutive days in 5.5 Gy fractionated doses (total: 27.5 Gy). Following irradiation, the minipigs were allowed 6 weeks of recovery for chronic irradiation skin changes to develop. After recovery, 1 cm × 1 cm full-thickness wounds were created in the irradiated fields. After wound creation, 100 μl of fibronectin hydrogel was topically applied on experimental wounds, and 100 μl of phosphate-buffered saline hydrogel was applied on control wounds. Wound photographs were taken at weekly time intervals to calculate the percentage of wound closure relative to original wound size. Tissues isolated from the wound areas were evaluated histologically for wound healing quality and analyzed for gene and protein levels of radiation injury mediators with quantitative RT-PCR (RT-qPCR) and ELISA. RESULTS: Wounds treated with fibronectin hydrogel demonstrated significantly faster wound closure and decreased scarring than wounds treated with phosphate-buffered saline hydrogel. By postoperative day 21, the mean percentage of wound area relative to original wound size was significantly higher in the control wounds (20.5 ± 2.6%) than in the fibronectin-treated wounds (4.3 + 0.9%). By postoperative day 28, the mean percentage of control wound area was 6.1 ± 2.7% while all fibronectin-treated wounds were fully healed. Picrosirius red staining demonstrated that the fibronectin-treated wounds had decreased total scar area (10.3 ± 2.3 mm2) compared with control wounds (37.7 ± 3.2 mm2). In addition, fibronectin hydrogel treatment was associated with decreased levels of radiation-induced inflammatory mediators, TGF-β1 and SMAD3. RT-qPCR of tissue collected from fibronectin-treated wounds had significantly lower mRNA levels of TGF-β1 (0.40 ± 0.07) compared with levels in control wounds (1.0 ± 0.10). Similarly, RT-qPCR data revealed that relative mRNA levels of SMAD3 were significantly lower in fibronectin-treated wounds (0.34 ± 0.08) than in control wounds (1.0 ± 0.18). Lastly, protein level correlation with ELISA found significantly lower TGF-β1 concentrations in fibronectin-treated wounds (2682 + 515.83 pg/mL) compared with control wounds (5245 + 700.08 pg/mL). CONCLUSIONS: Our novel hydrogel therapy functioned as a moisture-rich dressing and bioactive compound carrier. This dressing addresses irradiated skin fibronectin deficiency with topical glycoprotein supplementation, leading to improved rate and quality of chronic irradiated porcine wound healing.

Loss of ciliated cells and altered airway epithelial integrity in cystic fibrosis

BackgroundIn cystic fibrosis, the respiratory epithelium is the target tissue of both the genetic abnormality of the disease and of external aggressions, notably by pathogens (Pseudomonas aeruginosa). A detailed characterisation of the cystic fibrosis bronchial epithelium is however lacking, as most previous studies focused on the nasal epithelium or on cell lines. This study aimed to characterise the abnormal phenotype and epithelial-to-mesenchymal transition in cystic fibrosis bronchial epithelium and to evaluate in cell cultures whether abnormalities persist ex vivo. MethodsExplant lung tissues (n = 44) were assessed for bronchial epithelial cell phenotyping by immunostaining. Human bronchial epithelial cells were derived from basal cells isolated from cystic fibrosis patients or control donors and cultured in air-liquid interface for 2, 4 or 6 weeks. ResultsEnhanced mucin 5AC and decreased β-tubulin expression were observed in cystic fibrosis airways reflecting a decreased ciliated/goblet cell ratio, associated with increased number of vimentin-positive cells, indicating epithelial-to-mesenchymal transition process. These features were recapitulated in vitro, in cystic fibrosis-derived reconstituted epithelium. However, they were not induced by CFTR inhibition or Pseudomonas infection, and most abnormalities tended to disappear in long-term culture (6 weeks) except for increased fibronectin release, an epithelial-to-mesenchymal transition marker. ConclusionsThis study provides new insights into airway epithelial changes in cystic fibrosis, which are imprinted through an acquired mechanism that we could not relate to CFTR function.

High carbohydrate high fat diet causes arterial hypertension and histological changes in the aortic wall in aged rats: The involvement of connective tissue growth factors and fibronectin.

Age and diabetes are risk factors for arterial hypertension. However, the relationship between age, connective tissue growth factors, vascular aging and arterial hypertension while on a the high-carbohydrate high-fat diet (HCHFD) remains poorly understood. To estimate the relationship between humoral factors, the morphological changes of aorta and impaired blood pressure regulation under the influence of age and a HCHFD. A study was carried out in male Wistar rats, which were divided into the following groups: 1st (n=15) - naive young rats; 2nd (n=15) - young rats, exposed to HCHFD; 3rd (n=14) - naive old rats; 4th (n=12) - old rats exposed to HCHFD. The age of old rats was 540days, and young rats 150days at the end of the diet. HCHFD contained proteins 16%, fats 21%, carbohydrates 46%, including 17% fructose, 0.125% cholesterol, 90days. Blood pressure and body weight were measured weekly, carbohydrate metabolism, histological signs of changes in the aorta, serum transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), fibronectin, and endothelin-1 levels were determined one week after the onset of diet. The severity of arterial hypertension and its histological signs in the aortic wall was found to be most pronounced in elderly rats kept on a HCHFD. In young rats kept on a HCHFD, arterial hypertension was transient. An increase in systolic blood pressure has a positive correlation with the degree of obesity, serum fibronectin, and endothelin-1 content, and impaired carbohydrate metabolism. The rise in diastolic blood pressure has a positive correlation with the serum CTGF, endothelin-1, fibronectin levels and aortic wall thickness, and impaired carbohydrate metabolism. A rise in the serum concentration of fibronectin was also associated with increased endothelin-1, TGFβ and CTGF serum levels. This study indicated that an increase in blood pressure in old rats with a high-carbohydrate high-fat diet is due to a disturbance of a structure of the vascular wall, the release of fibronectin, which can occur under the influence of carbohydrate metabolism disorders, endothelin-1, TGFβ and CTGF.

5: 3D Transglutaminase Fibronectin Hydrogel Therapy Improves the Healing of Chronic Irradiated Porcine Skin Wounds

Purpose:Chronic irradiated wounds are characterized by a delayed and incomplete healing course. Currently, there are no therapies directed at the deficient or dysfunctional biology associated with cutaneous radiation injury. We have previously demonstrated that fibronectin is a key extracellular matrix glycoprotein known to be significantly downregulated in radiation-damaged skin. We further identified that an enzymatically crosslinked hydrogel is a suitable construct for incremental fibronectin release in vitro and in murine wound models. Our present objective was to investigate the design of this fibronectin hydrogel dressing for the treatment of irradiated wounds in the clinically relevant porcine irradiated wound model.Methods:We created a chronic irradiation skin injury model in female Yucatan minipigs. Two 1-month-old minipigs underwent irradiation of the right dorsolateral neck region for 5 consecutive days in 5.5 Gy fractionated doses for a total of 27.5 Gy. Following irradiation, the minipigs were allowed 6 weeks of recovery to enable chronic irradiation skin changes to develop. After recovery, nine 1 cm x 1 cm full-thickness wounds were created in the irradiated fields. After wound creation, 100 μl of fibronectin hydrogel was topically applied on experimental wounds and 100 μl of phosphate-buffered saline (PBS) hydrogel was applied on control wounds. Standardized wound photographs were taken at weekly time intervals to calculate the percentage of wound closure relative to original wound size. Tissues isolated from the wound areas were evaluated histologically for wound healing quality and analyzed for gene and protein levels of radiation injury mediators with quantitative RT-PCR and ELISA.Results:Wounds treated with fibronectin hydrogel demonstrated significantly faster wound closure and decreased scarring than wounds treated with PBS hydrogel. On postoperative day 21, the mean percentage of wound area relative to original wound size was significantly higher in the control wounds (21.3% ± 2.8%) than in the fibronectin-treated wounds (4.7 ± 1.0%). By the experimental endpoint on postoperative day 28, the mean percentage of control wound area was 6.8% ± 2.9% while all fibronectin-treated wounds were fully healed. Picrosirius red staining demonstrated that the fibronectin-treated wounds had decreased total scar area (9.9 ± 3.0 mm2) compared to control wounds (38.1 ± 3.6 mm2). In addition, fibronectin hydrogel treatment was associated with decreased levels of radiation-induced inflammatory mediators. RT-qPCR of samples from fibronectin-treated wounds had significantly lower mRNA levels of TGF-β1 (0.45 ± 0.09) compared to levels in control wounds (1 ± 0.13). Similarly, RT-qPCR data revealed that relative mRNA levels of SMAD3 were significantly lower in fibronectin-treated wounds (0.34 ± 0.11) than in control wounds (1 ± 0.43). Lastly, protein level correlation with ELISA identified significantly lower TGF-β1 concentrations in fibronectin-treated wounds (2682 + 515.83 pg/mL) compared to control wounds (5244.5 + 700.08 pg/mL).Conclusion:Hydrogel-facilitated delivery of fibronectin significantly improved the rate and quality of wound healing in a porcine chronic irradiation wound model. Thus, this novel mechanism of fibronectin supplementation demonstrates potential for treating these otherwise nonhealing wounds.

Protective effects of curcumin on bleomycin-induced changes in lung glycoproteins.

The present study investigated the therapeutic effect of curcumin on bleomycin (BLM)-induced alterations in glycoprotein components in the fibrotic lungs. Analysis of the bronchoalveolar lavage fluid (BALF) demonstrated increased fibronectin content at 3, 5, 7, and 14days after BLM administration. Similarly, lung tissue fibronectin content revealed a progressive increase at various times (days 3, 5, 7, 14, and 28) during the development of lung fibrosis. In addition, alveolar macrophage release of fibronectin was also elevated in BLM-treated rats. Analysis of carbohydrate moieties of glycoproteins revealed an increase in total hexose, fucose, sialic acid and hexosamine levels at 7, 14, and 28days after BLM treatment. Furthermore, the activities of lung glycosidases such as N-acetyl-β-D-glucosaminidase, β-glucosidase, β-galactosidase, and β-fucosidase in the fibrotic rats were elevated. Importantly, curcumin significantly inhibited the BLM-induced increases in BALF and lung fibronectin levels. Treatment of BLM rats with curcumin dramatically suppressed alveolar macrophage release of fibronectin. Curcumin also inhibited the increases in complex carbohydrates and glycosidases in the fibrotic lungs. These findings suggest that BLM-induced lung fibrosis is associated with accumulation of glycoproteins, and curcumin has the ability to suppress the enhanced deposition of glycoproteins in the fibrotic lung.

Ginsenoside Rg1-induced activation of astrocytes promotes functional recovery via the PI3K/Akt signaling pathway following spinal cord injury

AimsTo determine whether ginsenoside Rg1 is involved in scratch wound healing through altered expression of related molecules in astrocytes and improved functional recovery after spinal cord injury (SCI). Materials and methodsAstrocytes were isolated from rats, followed by Rg1 treatment. The wound healing test was performed to observe the scratch wound healing in different groups. The expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and components of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway were detected by western blot. Reverse transcription–polymerase chain reaction (RT-PCR) was used to measure the altered expression of laminin (LN) and fibronectin (FN). A revised Allen's method for the SCI model was performed, followed by Rg1 treatment. Then, functional scoring was conducted to evaluate the functional recovery. Hematoxylin-eosin (HE) staining showed changes in the void area. Finally, western blot assessed the expression of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). Key findingsRg1 mediated scratch wound healing through inducing an increased release of LN, FN, NGF, GDNF, and bFGF in vitro. Additionally, Rg1 activated the PI3K/Akt signaling pathway and promoted the functional recovery of hindlimb movement in rats. Furthermore, Rg1 significantly reduced the void area and downregulated the expression of GFAP and CSPGs. SignificanceRg1 not only enhanced the scratch wound repair in vitro through the release of astroglial neurotrophic factors, adhesion factors, and inhibitory factors, but it also improved the functional recovery in vivo following SCI.

Abstract 173: 3D Transglutaminase Fibronectin Hydrogel For The Treatment Of Skin Wounds

Purpose: Over the past decade, there has been a concerted effort to develop new artificial wound coverings that effectively treat major skin wounds. Hydrogels have emerged as novel drug delivery vehicles and wound dressing adjuncts, with a clinical dressing change frequency of 3 days given their unique moisture-conferring properties at the wound site. The topical application of fibronectin (FN) in a murine irradiated skin model has demonstrated improved wound healing by >20%. FN additionally has reported wound healing efficacy at the nanogram to microgram level. However, there is no reported application of hydrogel-facilitated FN delivery to promote wound healing. In this study, the in vitro and in vivo dynamics of 3D-transglutaminase-hydrogel (3DTgH) FN release were evaluated to characterize optimal parameters for the development of a novel wound dressing. Methods: 4.5A and 7.5A stiffness 3DTgH with 1 ug FN were constructed and plated in either PBS or collagenase type 2 (Col2) to allow gel digestion. Digested samples were assayed with FN ELISA. Additionally, full-thickness 8 mm punch biopsy dorsal skin wounds were created in 8-week-old FVB/NJ mice (n=22) weighing between 20 and 25 grams. Experimental mice (n=10) were treated with 4.5A stiffness 3DTgH incorporating 31.25 ug human FN (31.25 ul volume), and control mice (n=12) were treated with 4.5A stiffness 3DTgH incorporating 31.25 ul PBS. The total volume of each hydrogel treatment construct was 50 ul, and this suspension was applied topically onto the wound surface. For wound harvest on POD1, POD2, and POD3, residual hydrogel was carefully excised from the wound surface, and wounds were then harvested with 1 mm margins. Mouse tissue was homogenized and 100 ul samples were analyzed with human FN ELISA for pharmacokinetic (PK) analysis of FN release via hydrogel enzymatic digestion in vivo. Results: Unlike 3DTgH plated in PBS, 3DTgH plated in Col2 gradually dissolved. 4.5A 3DTgH maximally released FN on day 4 and 7.5A 3DTgH maximally released FN on day 13 with an overall slower rate of FN release. When compared with controls, mice treated with FN 3DTgH demonstrated sustained and increasing FN release via hydrogel after wound creation, with an average release of 125.31 ug FN on POD1 (p=0.0039), 141.91 ug on POD2 (p=0.0145), and 167.78 ug on POD3 (p=0.0001). Mice treated with control 3DTgH treatment did not display detectable levels of human FN via ELISA. Conclusion: 3DTgH is a suitable construct for gradual FN release in vitro and in vivo. Hydrogel constructs of differing stiffnesses demonstrate differential rates of FN release and thus have the potential to be tailored to wound healing environments for incremental FN delivery. As a result, hydrogel-based dressings incorporating FN have promise for clinical translation and sustained FN delivery in the treatment of skin wounds.

HMGB1 protein promotes glomerular mesangial matrix deposition via TLR2 in lupus nephritis.

Lupus nephritis (LN) is the most common complication of systemic lupus erythematosus. Patients with LN mostly die of sclerosing glomerulonephritis and renal failure. The inhibition of glomerular mesangial matrix deposition is an efficient method to restrict the progress of renal injury. By recognizing and binding extracellular and intracellular ligands, Toll-like receptor 2 (TLR2) contributes to the pathogenesis of most immune diseases. However, the relationship between TLR2 and LN is still unknown. Our previous studies confirmed that high-mobility group box 1 (HMGB1), an important ligand of TLR2, promotes the progression of LN by inducing the proliferation of glomerular mesangial cells. However, whether or not HMGB1 participates in the pathogenesis of glomerular mesangial matrix deposition in LN remains unknown. In this study, we observed the upregulated expression of TLR2 in the glomeruli of LN patients and MRL/lpr mice. The inhibition of either TLR2 or HMGB1 inhibited the release of fibronectin and the activation of the MyD88/NF-κB pathway in mesangial cells cultured with LN plasma. In addition, both TLR2- and HMGB1-deficient mice showed reduced 24 hr urine protein levels and improved glomerular histological changes and sclerosis levels. These results indicate that TLR2 regulates glomerular mesangial matrix deposition in LN through the activation of the MyD88/NF-κB pathway by binding to HMGB1.

514-P: Group 2 Innate Lymphoid Cells Promote Renal Fibrosis through the Production of Th2 Cytokines in Diabetic Kidney Disease

Aim: To explore the role of Group 2 innate lymphoid cells (ILC2s) in the pathogenesis of renal fibrosis in diabetic kidney disease (DKD). Methods: The proportion of ILC2s and ILC2s-producted Th2 cytokines (IL-4, IL-5, IL-13) in the peripheral blood of normal control subjects (NC) or patients with type 2 diabetes (DM), early diabetic kidney disease (DKD1), or late diabetic kidney disease (DKD2) were analyzed using flow cytometry and ELISA. Renal tubular epithelial cells (HK-2) were stimulated with IL-4 or IL-13 in the presence or absence of high glucose and the expression and release of transforming growth factor-β1 (TGF-β1) and fibronectin (FN) was analyzed using qPCR and ELISA. Results: The proportion of ILC2s and the level of IL-4, IL-5, and IL-13 were significantly increased with the severity of DKD (P<0.05). The expression and release of TGF-β1 and FN were significantly upregulated by IL-4 or IL-13 in HK-2 cells (P<0.05). Compared with high glucose stimulation alone, treatment with IL-4 or IL-13 combined with high glucose significantly stimulated HK-2 cells to increase expression of FN and TGF-β1 (P<0.05). Conclusions: ILC2s may promote the development and progression of diabetic kidney disease by secreting Th2 cytokines (IL-4 and IL-13) and promoting renal fibrosis. Disclosure C. Liu: None. L. Zhou: None. C. Gao: None. T. Zhang: None. M. Guo: None. W. Huang: None. Z. Jiang: None. J.Y. Ding: None. Y. Xu: None.

The Induction of Autoimmune Arthritis and Sex Differences in Mice Impact the Lung Inflammatory Response to Repetitive Inhalant Organic Dust Extract Exposures

Inflammatory lung diseases are often associated with systemic autoimmunity, and sex differences have been independently described. Men are at increased risk of occupational exposure-associated rheumatoid arthritis (RA) and extra-articular manifestations. Because agriculture work is associated with both chronic lung disease and RA, we sought to determine whether induction of autoimmune arthritis and sex influence airway response to organic dust extract (ODE). Arthritis prone, DBA/1J male and female mice (8 week), were treated daily with ODE or saline for 5 weeks with collagen-induced arthritis (CIA) induced on days 1 and 21. Treatment groups included Sham (saline injection/saline inhalation), CIA (CIA/saline), ODE (saline/ODE); CIA+ODE (CIA/ODE). Bronchoalveolar lavage fluids, lung tissues, serum, and bones were collected. ODE induced airway neutrophil influx, increases in inflammatory mediators (TNF-α, IL-6, CXCL1, CXCL2), release of fibronectin (but not hyaluronan), and increased lung collagen staining as compared to sham. Co-exposure (CIA+ODE) resulted in reduced neutrophil and inflammatory mediator responses as compared to ODE alone, but lung tissue levels of hyaluronan, fibronectin and collagen staining were either augmented or remained increased in CIA+ODE. Arthritis inflammatory scores and serum anti-cyclic citrullinated peptide antibody were greatest in CIA+ODE. Compared to male mice, female mice demonstrated reduced ODE-induced mediator release, hyaluronan and fibronectin as well as lower arthritis scores and serum autoantibody levels with co-exposure. Autoimmune arthritis induction modulates the lung response to inhalant ODE exposure, impacting the inflammatory-interstitial disease process. Congruent with epidemiologic findings, male mice were most susceptible to lung and articular disease resulting from ODE.

Functionalized Scaffold for in Situ Efficient Gene Transfection of Mesenchymal Stem Cells Spheroids toward Chondrogenesis.

Multicellular mesenchymal stem cell (MSC) spheroids possess enhanced chondrogenesis ability and limited fibrosis, exhibiting advantage toward hyaline-like cartilage regeneration. However, because of the limited cell surfaces in spheroid exposed to DNA/vector, it is difficult to realize efficient gene transfection, most of which highly rely on cell-substrate interaction. Here, we report a poly(l-glutamic acid)-based porous scaffold with tunable inner surfaces that can sequentially realize cell-scaffold attachment and detachment, as well as the followed in situ spheroid formation. The attachment and detachment of cells from scaffold is achieved by the capture and release of fibronectin (Fn) via reversible imine linkage between aromatic aldehyde groups of scaffold and amino groups of Fn. Together with N, N, N-trimethyl chitosan chloride condensing plasmid DNA encoding transforming growth factor-β1 (pDNA-TGF-β1), cell attachment realizes efficient surface-mediated gene transfection. Conversion of scaffold stiffness can affect the adhesion shape of cells. Stiffer scaffold reinforces the adhesion, leading to the amplification of peripheral focal adhesions and the promotion of cell spreading, as well as the promotion of gene transfection efficiency. After cellular detachment from the scaffold via lysine treatment, the subsequent spheroid formation with extensive cell-cell interaction up-regulates the corresponding protein expression with a prolonged term. With the induction effect of the expressed TGF-β1, significantly enhanced chondrogenesis of MSCs in spheroids is achieved at 10 d in vitro. Well-regenerated cartilage at 8 weeks in vivo indicates that the present gene transfection system is a platform that can be potentially applied toward cartilage tissue engineering.

Ribemansides A and B, TRPC6 Inhibitors from Ribes manshuricum That Suppress TGF-β1-Induced Fibrogenesis in HK-2 Cells.

Two new acylated β-hydroxynitrile glycosides, ribemansides A (1) and B (2), were isolated from the aerial parts of Ribes manshuricum. Their structures were elucidated by comprehensive spectroscopic analysis. Ribemansides A and B inhibited transforming growth factor β1 (TGF-β1)-induced expression of α-smooth muscle actin, fibronectin release, and changes in cell morphology in the human proximal tubular epithelial cell line (human kidney-2, HK-2). Further biological evaluation demonstrated that both 1 and 2 inhibit the activity of canonical transient receptor potential cation channel 6 (TRPC6), with IC50 values of 24.5 and 25.6 μM, respectively. The antifibrogenic effect of these compounds appears to be mediated through TRPC6 inhibition, since the TRPC6 inhibitor, SAR7334, also suppressed TGF-β1-induced fibrogenesis in HK-2 cells.

Contribution of activated beta3 integrin in the PDI release from endothelial cells

Protein disulfide isomerase (PDI) is an abundant reticulum endoplasmic protein but also acts as an important functional regulator of some extracellular surface proteins. Recent studies suggest that PDI plays a role in integrin activation and thrombus formation. The aim of this study was to examine whether activation of integrin is the first stage leading to release of PDI from the subcellular compartments of endothelial cells to extracellular space. Our results show that endothelial cells which adhere to fibronectin or fibrinogen release significantly more PDI than those which adhere to poly-L-lysine. Cells treated with RGD peptide, Src and FAK kinase inhibitors and anti alphaVbeta3 antibody display lower PDI secretion. The destruction of the actin cytoskeleton of endothelial cells by cytochalasin D inhibits PDI release. When the endothelial cells adhere to fibrinogen or fibronectin, PDI and alphaVbeta3 gain free thiol groups. Our data suggest that upon activation of integrins, PDI is released from endothelial cells and forms a disulfide bond complex with alphaVbeta3 integrin.

Reduced microRNA-503 expression augments lung fibroblast VEGF production in chronic obstructive pulmonary disease.

Alterations in microRNA (miRNA) expression may contribute to COPD pathogenesis. In COPD, lung fibroblast repair functions are altered in multiple ways, including extracellular mediator release. Our prior study revealed miR-503 expression is decreased in COPD lung fibroblasts, although the exact role played by miR-503 is undetermined. The current study examined a role of miR-503 in cytokine, growth factor and fibronectin production by lung fibroblasts from patients with and without COPD. Primary adult lung fibroblasts were isolated from patients with or without COPD. MiR-503 expression and interleukin (IL)-6, -8, PGE2, HGF, KGF, VEGF and fibronectin release were examined with or without inflammatory cytokines, IL-1β and tumor necrosis factor (TNF)-α. MiR-503 expression was decreased in COPD lung fibroblasts. The expression of miR-503 was positively correlated with %FVC, %FEV1, and %DLco as well as IL-6, -8, PGE2, HGF, KGF, and VEGF in the absence or presence of IL-1ß/TNF-α. In addition, IL-8 and VEGF release from COPD lung fibroblasts were increased compared to those from control. Exogenous miR-503 inhibited VEGF release from primary adult and fetal lung fibroblasts but not IL-8 release. As expected, COPD fibroblasts proliferated more slowly than control fibroblasts. MiR-503 did not affect proliferation of either control or COPD lung fibroblasts. MiR-503 inhibition of VEGF protein production and mRNA was mediated by direct binding to the 3’ untranslated region of VEGF mRNA. Endogenous miR-503 was differently regulated by exogenous stimulants associated with COPD pathogenesis, including IL-1ß/TNF-α, TGF-ß1 and PGE2. Endogenous miR-503 inhibition augmented VEGF release by IL-1ß/TNF-α and TGF-ß1 but not by PGE2, demonstrating selectivity of miR-503 regulation of VEGF. In conclusions, reduced miR-503 augments VEGF release from lung fibroblasts from patients with COPD. Since VEGF contributes to disturbed vasculature in COPD, altered miR-503 production might play a role in modulating fibroblast-mediated vascular homeostasis in COPD.

KATP channels in high glucose-induced rat mesangial cell proliferation and release of MMP-2 and fibronectin

ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. The purpose of this study was to determine if KATP channels play a role in diabetic nephropathy (DN). In the present study, functional expression of the KATP channel was examined in rat mesangial cells with or without high glucose (HG) stimulation. The mesangial cell proliferation and the release of matrix metalloproteinase (MMP)-2 and fibronectin in response to high glucose with a selective opener of KATP (diazoxide, DZX), or with a selective inhibitor of KATP (5-hydroxydecanoate, 5-HD) were also measured. The cell proliferation was observed using Cell Counting Kit-8 assay, and the mRNA expressions of KATP subunit, including Kir6.1, Kir6.2, sulfonylurea receptor 1 (SUR1), SUR2A and SUR2B, were assessed using quantitative real-time PCR. MMP-2 and fibronectin release was measured by ELISA. The present study clarified expression of SUR subunit of KATP in plasma. HG treatment could cause increased cell proliferation and release of MMP-2 and fibronectin in a dose-dependent manner. HG also significantly decreased the expression of Kir6.1, SUR2A and SUR2B. Pretreatment of DZX markedly decreased the expression of SUR1, SUR2A and SUR2B, but had no effect on Kir6.1 expression compared with HG alone, while these changes were inhibited by 5-HD pretreatment. Moreover, DZX also inhibited cell proliferation and release of MMP-2 and fibronectin in HG-induced rat mesangial cells, and that was corrected by 5-HD. These data suggest that HG stimulates mesangial cell proliferation and cellular matrix release via inhibiting KATP channel activity, leading us to propose that KATP channel dysfunction may be involved in the development of DN.

Inflammasome activators induce fibronectin expression and release in macrophages.

Extracellular fibronectin (Fn) can activate pro-inflammatory pathways and serves as an endogenous danger signalling molecule; thus, it has been suggested as a biomarker for several diseases. In the present study, we found that pathogen-derived activators of the inflammasomes induce the expression and secretion of Fn in macrophages through a mechanism involving adenosine triphosphate and caspase-1 activation. We also found that plasma Fn induces caspase-1 activation and cell death in macrophages, epithelial cells, and fibroblasts. Together, these results indicate that Fn plays a critical role in inflammasome-activated cells by amplifying caspase-1 activation and inducing inflammatory cell death.