The Wound Healing of Autologous Regenerative Factor on Recurrent Benign Airway Stenosis: A Canine Experimental and Pilot Study.

INTRODUCTION: Benign airway stenosis (BAS) is a severe pathologic condition. Complex stenosis has a high recurrence rate and requires repeated bronchoscopic interventions for achieving optimal control, leading to recurrent BAS (RBAS) due to intraluminal granulation. METHODS: This study explored the potential of autologous regenerative factor (ARF) for treating RBAS using a post-intubation tracheal stenosis canine model. Bronchoscopic follow-ups were conducted, and RNA-seq analysis of airway tissue was performed. A clinical study was also initiated involving 17 patients with recurrent airway stenosis. RESULTS: In the animal model, ARF demonstrated significant effectiveness in preventing further collapse of the injured airway, maintaining airway patency and promoting tissue regeneration. RNA-seq results showed differential gene expression, signifying alterations in cellular components and signaling pathways. The clinical study found that ARF treatment was well-tolerated by patients with no severe adverse events requiring hospitalization. ARF treatment yielded a high response rate, especially for post-intubation tracheal stenosis and idiopathic tracheal stenosis patients. CONCLUSION: The study concludes that ARF presents a promising, effective, and less-invasive method for treating RBAS. ARF has shown potential in prolonging the intermittent period and reducing treatment failure in patients with recurrent tracheal stenosis by facilitating tracheal mucosal wound repair and ameliorating tracheal fibrosis. This novel approach could significantly impact future clinical applications.

The synergic inhibitory effects of dark tea (Camellia sinensis) extract and p38 inhibition on the growth of pancreatic cancer cells.

Background: Dark tea is one of the most popular types of Chinese tea, which has been reported to exhibit anti-obesity, anti-oxidation and antitumor activities in according human cell lines. In terms of tumorigenesis, the systemic study of the physiological effect of specific fraction of dark tea and the relevant molecular mechanism warrant more attention. Methods: Dark tea was firstly isolated through solvent extraction method. Dissolved ethyl acetate extract was further fractioned by elution with various concentration of ethyl alcohol. The cytotoxicity effect of dark tea on cell proliferation was evaluated by CCK8 assay in HPDE human normal pancreatic duct epithelial cells, SW1990 and PANC-1 human pancreatic cancer cells, and SW1116 human colorectal cancer cells. Immunoblotting and flow cytometry analysis were utilized to examine the status of protein and reactive oxygen species respectively. Gene expression profile was analyzed by cDNA microarray and real-time PCR. The plasmid for ID1 expression was stably transfected into SW1990 cells for relevant functional analysis. The effect of dark tea extract on tumorigenesis in vivo was studied in xenograft tumor model. Results: Water eluate fraction of the ethyl acetate extract from dark tea inhibited the growth of SW1990, PANC-1 and SW1116 cells more efficiently compared with that in HPDE cells. Meanwhile, p38 activity was increased and AKT activity was dropped in cancer cells with dark tea extract treatment. Further functional analyses indicated that water eluate fraction and p38 inhibitor treatment exerted a synergic inhibitory effect on cancer cells growth, which was related to their suppressive effect on expression level of ID1 (inhibitor of differentiation protein 1), which was highly expressed in cancer cells. The analysis utilizing xenograft tumor model further indicated water eluate fraction exhibited a significantly inhibitory effect on tumorigenesis. Conclusion: Based on the sequential extraction procedure, our results reveal the inhibitory effect of water eluate fraction of the ethyl acetate extract from dark tea and its synergistic effect with p38 inhibition on the growth of pancreatic cancer cells, in which ID1 is identified as a downstream effector. This sheds insights into the physiological relevance of specific fraction of dark tea to tumorigenesis in pancreatic cancer.

Cadmium favors F-actin depolymerization in rat renal mesangial cells by site-specific, disulfide-based dimerization of the CAP1 protein.

Cadmium is a toxic metal that produces oxidative stress and has been shown to disrupt the actin cytoskeleton in rat renal mesangial cells (RMC). In a survey of proteins that might undergo Cd2+-dependent disulfide crosslinking, we identified the adenylyl cyclase-associated protein, CAP1, as undergoing a dimerization in response to Cd2+ (5-40 µM) that was sensitive to disulfide reducing agents, was reproduced by the disulfide crosslinking agent diamide, and was shown by site-directed mutagenesis to involve the Cys29 residue of the protein. Reactive oxygen species are not involved in the thiol oxidation, and glutathione modulates background levels of dimer. CAP1 is known to enhance cofilin's F-actin severing activity through binding to F-actin and cofilin. F-actin sedimentation and GST-cofilin pulldown studies of CAP1 demonstrated enrichment of the CAP1 dimer's association with cofilin, and in the cofilin-F-actin pellet, suggesting that Cd2+-induced dimer increases the formation of a CAP1-cofilin-F-actin complex. Both siRNA-based silencing of CAP1 and overexpression of a CAP1 mutant lacking Cys29 (and therefore, incapable of dimerization in response to Cd2+) increased RMC viability and provided some protection of F-actin structures against Cd2+. It is concluded that Cd2+ brings about disruption of the RMC cytoskeleton in part through formation of a CAP1 dimer that increases recruitment of cofilin to F-actin filaments.

Cadmium-induced aggregation of iron regulatory protein-1.

Iron regulatory protein-1 (IRP-1) is central to regulation of iron homeostasis, and has been shown to be sensitive to Cd(2+) in vitro. Although Cd(2+) induces disulfide-bond formation in many proteins, the critical cysteine residues for iron binding in IRP-1 were shown not to be involved in Cd-induced IRP-1 aggregation in vitro. Here we show that Cd(2+) causes polymerization and aggregation of IRP-1 in vitro and in vivo, and decreases in a dose-dependent manner both its RNA-binding and aconitase enzymatic activities, as well as its cytosolic expression. We have used two-dimensional electrophoresis to demonstrate thiol-dependent self-association of purified recombinant IRP-1 treated with Cd(2+), as well as self-association in Cd(2+)-exposed mesangial cells. Circular dichroism spectra confirm significant conformational changes in the purified protein upon Cd(2+) exposure. Following Cd(2+) treatment, there is increased translocation of inactive IRP-1 to the actin cytoskeletal fraction, and this translocation is diminished by both antioxidant (BHA) treatment and inhibition of CaMK-II. These changes differ from those elicited by manipulation of iron levels. Cadmium-induced translocation of proteins to cellular compartments, and particularly to the cytoskeleton, is becoming a recognized event in Cd(2+) toxicity. Polymer-dependent translocation of IRP-1 in Cd(2+)-exposed cells may underlie effects of Cd(2+) on iron homeostasis.

Cadmium-induced glutathionylation of actin occurs through a ROS-independent mechanism: implications for cytoskeletal integrity.

Cadmium disrupts the actin cytoskeleton in rat mesangial cells, and we have previously shown that this involves a complex interplay involving activation of kinase signaling, protein translocation, and disruption of focal adhesions. Here we investigate the role that glutathionylation of actin plays in Cd(2+)-associated cytoskeletal reorganization. Low concentrations of Cd(2+) (0.5-2 µM) caused an increase in actin glutathionylation by 6h, whereas at higher concentrations glutathionylation remained at basal levels. Although oxidation with diamide increased glutathionylation, reactive oxygen species (ROS) were not involved in the Cd(2+)-dependent effect, as only Cd(2+) concentrations above 2 µM were sufficient to increase ROS. However, low [Cd(2+)] increased total glutathione levels without affecting the ratio of reduced/oxidized glutathione, and inhibition of glutathione synthesis suppressed actin glutathionylation. Cadmium increased the activity of the enzyme glutaredoxin, which influences the equilibrium between glutathionylated and deglutathionylated proteins and thus may influence levels of glutathionylated actin. Together these observations show that cadmium-dependent effects on actin glutathionylation are affected by glutathione metabolism and not by direct effects of ROS on thiol chemistry. In vitro polymerization assays with glutathionylated actin show a decreased rate of polymerization. In contrast, immunofluorescence of cytoskeletal structure in intact cells suggests that increases in actin glutathionylation accompanying increased glutathione levels occurring under low Cd(2+) exposure are protective in vivo, with cytoskeletal disruption ensuing only when higher Cd(2+) concentrations increase ROS levels and prevent an increase in actin-glutathione conjugates.

Low-concentration heparin suppresses ionomycin-activated CAMK-II/EGF receptor- and ERK-mediated signaling in mesangial cells.

Heparin and endogenous heparinoids inhibit the proliferation of smooth muscle cells, including renal mesangial cells; multiple effects on signaling pathways are well established, including effects on PKC, Erk, and CaMK-II. Many studies have used heparin at concentrations of 100 microg/ml or higher, whereas endogenous concentrations of heparinoids are much lower. Here we report the effects of low-concentration (1 microg/ml) heparin on activation of several kinases and subsequent induction of the c-fos gene in mesangial cells in response to the calcium ionophore, ionomycin, in the absence of serum factors. Ionomycin rapidly increases the phosphorylation of CaMK-II (by 30 s), and subsequently of the EGF receptor (EGFR), c-Src, and Erk 1/2. Low-dose heparin suppresses the ionomycin-dependent phosphorylation of EGFR, c-Src, and Erk 1/2, but not of CaMK-II, whereas inhibition of activated CaMK-II reduces phosphorylation of EGFR, c-Src, and Erk. Our data support a mechanism whereby heparin acts at the cell surface to suppress downstream targets of CaMK-II, including EGFR, leading in turn to a decrease in Erk- (but not c-Src-) dependent induction of c-fos.

Pleiotropic effects of cadmium in mesangial cells.

The mesangial cell of the renal glomerulus is exposed to circulating toxic substances and is a target involved in the glomerular component of chronic occupational and environmental exposure to cadmium. We review evidence for the involvement of cadmium in mesangial cell pathology, including effects on cell signaling, oncogene expression, and cell death. Previously we have shown that cadmium can inhibit apoptosis initiated through both the extrinsic (death ligand receptor) and intrinsic (mitochondrial) pathways, whereas exposure of mesangial cells to 10 microM CdCl(2) for 6 h initiates caspase-independent cell death through both apoptotic and apoptotic-like (annexin V positive, propidium iodide staining) mechanisms. Apoptotic death is dependent upon activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II). In the present study we show that low level exposure of mesangial cells to Cd(2+) (0.5 microM) initiates cell survival signals including PI3 kinase/Akt signaling, also dependent on CaMK-II, that are eventually overcome resulting in caspase-dependent cell death. These studies underscore the roles of cell signaling in various modes of cell death, and in particular the central role of CaMK-II in cadmium toxicology of the mesangial cell.

Cadmium inhibits both intrinsic and extrinsic apoptotic pathways in renal mesangial cells.

Cadmium is a potent nephrotoxin that has been shown to induce apoptosis in some cells but also to prevent it under certain circumstances. In several clinical situations and experimental models of injury to the renal glomerulus, pathological proliferation of mesangial cells is followed by resolution involving mesangial cell apoptosis. We investigated the effects of Cd(2+) on rat mesangial cells induced to undergo apoptosis through either the extrinsic receptor-mediated pathway or the intrinsic mitochondrial-dependent pathway. Camptothecin initiated the intrinsic pathway with activation of caspase-9 and caspase-dependent cleavage of procaspase-3. Tumor necrosis factor-alpha (TNF-alpha) initiated caspase-8 activity and cleavage of pro-caspase-3 at the convergence point of the two pathways. However, pro-caspase-8 levels were low, and caspase-9 was also activated in response to TNF-alpha, characteristic of what have been termed type II cells. With both TNF-alpha and camptothecin, concurrent exposure to 10 microM CdCl(2) suppressed DNA laddering, nuclear condensation, and pro-caspase-3 cleavage. It also decreased activity of both caspase-8 and caspase-9, prevented caspase-8-dependent cleavage of the proapoptotic factor Bid, and suppressed release of cytochrome c from mitochondria. At this 10-microM concentration, Cd(2+) was unique among a number of metal ions in preventing DNA fragmentation. We conclude that Cd(2+) is anti-apoptotic in rat mesangial cells, acting by a mechanism that may involve general caspase inhibition. This may have consequences for the resolution of nephritis in situations of mesangial cell hyperproliferation.

Ca(2+)/calmodulin-dependent protein kinase II inhibition by heparin in mesangial cells.

Heparin exerts an antiproliferative effect in smooth muscle cells, and the Ca(2+)/calmodulin-dependent protein kinase (CaMK) signaling pathway is heparin sensitive. Here, we report that transfection with a truncated 326-amino acid fragment of CaMK-IIalpha increases basal activity of CaMK-II in mesangial cells. Ionomycin increased CaMK-II activity in both transfected and untransfected cells, with a concomitant increase in activated Ca(2+)/calmodulin. Heparin (1 microg/ml), but not chondroitin or dermatan sulfate, significantly attenuated both serum- or ionomycin-induced CaMK-II activity, and attendant c-fos mRNA expression, but did not affect upstream Ca(2+)/calmodulin. Autophosphorylation of Thr286 generates an autonomously active CaMK-II. Both serum and ionomycin increased phosphorylation at this site and increased CaMK-II activity in antiphosphothreonine immunoprecipitates. Heparin (1 microg/ml) did not inhibit phosphorylation of Thr286 (although much higher concentrations did). Replacement of Thr286 with Asp produces a constitutively active mutant that was insensitive to ionomycin but was inhibited by heparin maximally at 1 microg/ml. These results suggest that heparin at physiological concentrations acts at or downstream of CaMK-II to suppress its activity independent of an effect on autophosphorylation.

Suppression of mitogen-activated protein kinase phosphatase-1 (MKP-1) by heparin in vascular smooth muscle cells.

Heparin inhibits vascular smooth muscle cell (VSMC) proliferation, but mechanisms remain elusive. Because heparin inhibits signaling through multiple kinase cascades, we investigated the possibility that phosphatases could be involved. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was the predominant MKP detected in VSMC lines. MKP-1 protein was increased by serum stimulation of quiescent cells, and this increase was diminished by heparin (1 microg/mL). Increased MKP-1 expression was dependent on the mitogen-activated protein kinase, Erk. Decreased Erk activity in the presence of heparin preceded, and may account for, decreased MKP-1. The antimitogenic effects of heparin are therefore unlikely to act through a shift in the kinase/phosphatase balance, but rather through direct kinase suppression. However, because MKP-1 is known to cause an increase in activity of kinases upstream of Erk, that may signal through additional pathways, the decrease in MKP-1 activity may paradoxically enhance heparin's antiproliferative effects. VSMC selected to grow in the presence of heparin express decreased levels of MKP-1 that are unresponsive to heparin, and Erk activity becomes unresponsive to heparin in one cell line. We conclude that phosphatase activation is not a direct mechanism of suppression of multiple kinase cascades by heparin.