Zhenxiang Zhang

Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells

BACKGROUND Pulmonary emphysema is characterized by loss of alveolar structures. We have found that bone marrow (BM) mesenchymal stem cell (MSC) transplantation ameliorates papain-induced pulmonary emphysema. However, the underlying mechanism is not completely understood. It has been shown that blocking the vascular endothelial growth factor (VEGF) signaling pathway leads to apoptosis of lung cells and pulmonary emphysema, and MSC are capable of secreting VEGF. We hypothesized that MSC transplantation may have a protective effect on pulmonary emphysema by increasing VEGF-A expression and inhibiting apoptosis of lung cells. METHODS We examined the morphology and expression of VEGF-A in rat lung after papain treatment and MSC transplantation. We also used a co-culture system in which MSC and cells prepared from papain-treated lungs or control lungs were cultured together. The levels of VEGF-A in cells and culture medium were determined, and apoptosis of cultured lung cells was evaluated. RESULTS VEGF-A expression in rat lungs was decreased after papain treatment, which was partly rescued by MSC transplantation. MSC production of VEGF-A was increased when MSC were co-cultured with cells prepared from papain-treated lungs. Furthermore, the apoptosis of papain-treated lung cells was inhibited when co-cultured with MSC. The induction of MSC production of VEGF-A by papain-treated lung cells was inhibited by adding anti-tumor necrosis factor (TNF)-alpha antibody to the medium. CONCLUSIONS The protective effect of MSC transplantation on pulmonary emphysema may be partly mediated by increasing VEGF-A expression and inhibiting the apoptosis of lung cells. TNF-alpha released from papain-treated lung cells induces MSC to secret VEGF-A.

N -Acetyl- L -cysteine and pyrrolidine dithiocarbamate inhibited nuclear factor-kappaB activation in alveolar macrophages by different mechanisms

AbstractAim:To study the effects of N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) on the phosphorylation of IκB kinase (IKK)β, IKKα, and IκBα in alveolar macrophages (AM), and to explore the pharmacological mechanisms of NAC and PDTC as inhibitors of NF-κB activation.Methods:AM were collected from bronchoalveolar lavage fluid from the patients with chronic obstructive pulmonary disease. The AM were incubated for 1.5 h with NAC and PDTC, and then stimulated for 90 min by either tumor necrosis factor (TNF)-α or interleukin (IL)-1. Western blotting was used to detect the protein phosphorylation levels of IKKβ, IKKα, and IκBα. NF-κB activity was analyzed by using an electrophoretic mobility shift assay.Results:NAC inhibited the phosphorylation of IKKβ, IKKα, and IκBα induced by TNF-α, but had no effect on the phosphorylation of IKKβ, IKKα and IκBα induced by IL-1. PDTC did not inhibit the phosphorylation of IκBα induced by TNF-α or IL-1. Similarly, NAC inhibited the activation of NF-κB induced by TNF-α, but had no effect on the activation of NF-κB induced by IL-1. PDTC significantly inhibited the activation of NF-κB induced by TNF-α and IL-1. The electrophoretic mobility shift assay also showed that PDTC and NAC do not directly inhibit NF-κB DNA binding activity in vitro.Conclusion:PDTC prevents the degradation of IκBα via the ubiquitylation-proteasome proteolytic pathway. NAC can inhibit the processes upstream of IKK activation induced by TNF-α, which results in the decline of NF-κB activity.

Intelectin is required for IL-13-induced monocyte chemotactic protein-1 and -3 expression in lung epithelial cells and promotes allergic airway inflammation

Asthma is characterized by airway inflammation, mucus overproduction, airway hyperreactivity, and peribronchial fibrosis. Intelectin has been shown to be increased in airway epithelium of asthmatics. However, the role of intelectin in the pathogenesis of asthma is unknown. Airway epithelial cells can secrete chemokines such as monocyte chemotactic protein (MCP)-1 and -3 that play crucial roles in asthmatic airway inflammation. We hypothesized that intelectin plays a role in allergic airway inflammation by regulating chemokine expression. In a mouse allergic asthma model, we found that mRNA expression of intelectin-2 as well as MCP-1 and -3 in mouse lung was increased very early (within 2 h) after allergen challenge. Expression of intelectin protein was localized to mucous cells in airway epithelium. Treatment of MLE12 mouse lung epithelial cells with interleukin IL-13, a critical mediator of allergic airway disease, induced expression of intelectin-1 and -2 as well as MCP-1 and -3. When IL-13-induced intelectin-1 and -2 expression was inhibited by RNA interference, IL-13-induced extracellular signal-regulated kinase 1/2 phosphorylation and MCP-1 and -3 production by MLE12 cells was inhibited. Furthermore, inhibition of intelectin expression by airway transfection with shRNA targeting intelectin-1 and -2 attenuated allergen-induced airway inflammation. We conclude that intelectin, a molecule expressed by airway epithelial cells and upregulated in asthma, is required for IL-13-induced MCP-1 and -3 production in mouse lung epithelial cells and contributes to allergic airway inflammation.

Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease

BackgroundStem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats.MethodsThe MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay.ResultsrAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions.ConclusionsVacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.

Up-regulation of cyclin D1 expression in asthma serum-sensitized human airway smooth muscle promotes proliferation via protein kinase Cα

ABSTRACT Abnormal hypertrophy and hyperplasia of airway smooth muscle cells play an important role in airway remodeling in chronic asthma. The authors’ previous studies have indicated that protein kinase Cα (PKCα) is involved in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). However, the underlying mechanisms remain unknown. Here, the authors examined the possible role of the α isoform of PKC in the control of cyclin D1 expression, using HASMCs passively sensitized on human atopic asthmatic serum as a model system. Cultured HASMCs were passively sensitized with serum from atopic asthmatic patients. Cell proliferation was measured by [3H]thymidine incorporation and an MTT assay. Cell cycle status was analyzed by flow cytometry. The mRNA and protein expression profiles of cyclin D1 were measured by reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blotting, respectively. Furthermore, the authors assessed the role of cyclin D1 in PKCα-induced HASMC proliferation by transfection with a recombinant cyclin D1 antisense construct. The activation of PKCα with phorbol myristate acetate (PMA), a PKC activator, up-regulated cyclin D1 expression and increased the proliferation of passively sensitized HASMCs. This effect was significantly decreased by specific inhibition of PKCα with Go6976. In addition, the authors showed that transfection with antisense cyclin D1 abolished PMA-induced G1/S progression and HASMC proliferation. These results demonstrate that PKCα promotes the proliferation of HASMCs sensitized with atopic asthmatic serum via up-regulation of cyclin D1 expression.

ERK1/2 signaling pathway modulates the airway smooth muscle cell phenotype in the rat model of chronic asthma.

Background: It has been demonstrated that the phenotypic modulation of airway smooth muscle cells (ASMCs) is important to the pathogenesis of airway remodeling in chronic asthma. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is one of the most important transduction pathways involved in the process of asthma; however, its role in the phenotypic transition of ASMCs remains unclear. Objectives: To examine the role of ERK1/2 in the phenotypic modulation of ASMCs in the rat model of chronic asthma. Methods: Bronchial smooth muscle strips were cultured in vitro in the presence of the ERK1/2 agonist epidermal growth factor or/and the MEK inhibitor PD98059. The phenotype of ASMCs was determined by observing these cells under an electron microscope and analyzing expression of phenotypic markers (smooth muscle α-actin for the contractile phenotype and osteopontin for the synthetic) by using Western blot and reverse-transcriptase polymerase chain reaction, respectively. Results: The phenotype of the ASMCs from the chronic asthmatic rats changed from the contractile type to the synthetic type with synthetic organelles abundantly gathered around the nucleus and altered expression of phenotypic markers. ERK1/2 was strongly expressed in the ASMCs of the chronic asthmatic rats and its activation by epidermal growth factor excessively promoted the synthetic function of ASMCs; the MEK inhibitor PD98059, however, reversed this phenotypic change in the ASMCs. Conclusions: Our results reveal a key role of the ERK1/2 signaling pathway in the phenotypic modulation of ASMCs in chronic asthmatic rats, indicating that specific inhibition of ERK1/2 in ASMCs may be therapeutically valuable in the control of airway remodeling in chronic asthma.

5-Hydroxydecanoate inhibits proliferation of hypoxic human pulmonary artery smooth muscle cells by blocking mitochondrial KATP channels

AbstractAim:To study the effect of 5-hydroxydecanoate (5-HD) on the proliferation of 24 h hypoxic human pulmonary artery smooth muscle cells (HPASMC) and to explore the pharmacological mechanisms of 5-HD as an inhibitor of mitochondrial membrane ATP-sensitive potassium channel activation.Methods:Normoxic or hy-poxic HPASMC in culture were stimulated by either diazoxide or 5-HD for 24 h. The proliferation of HPASMC was examined by 3- (4,5-dimethyl-2-thiazol-yl) -2,5-diphenyl- 2H-tetrazolium bromide (MTT) assay and proliferating cell nuclear antigen (PCNA) immunohistochemistry staining. The apoptosis of HPASMC was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and flow cytometric analysis. The relative changes in mitochondrial membrane potential (ΔΨm) were measured using the rhodamine fluorescence (R-123) technique.Results:Both hypoxia and diazoxide stimulation increased ΔΨm value measured by the absorbance of MTT, PCNA-positive staining and decreased TUNEL-positive staining and apoptotic cells in HPASMC. Hypoxia and the concomitant stimulation of diazoxide obviously enhanced the effects of hypoxia or diazoxide alone. 5-HD significantly attenuated the effects in each of the above conditions. Additionally, 5-HD partially inhibited the effect of hypoxia on R-123 fluorescence intensity in HPASMC.Conclusion:5-HD can inhibit the proliferation of hypoxic HPASMC by blocking mitochondrial KATP channels.

The alteration and significance of surfactant protein A in rats chronically exposed to cigarette smoke

In order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 ± 0.05) was lower than that of the N group (0.72±0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522±0.0512) was significantly lower than that of the N group (0.4432±0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.SummaryIn order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 ± 0.05) was lower than that of the N group (0.72±0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522±0.0512) was significantly lower than that of the N group (0.4432±0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.

The effects of protein kinase C (PKC) on the tension of normal and passively sensitized human airway smooth muscle and the activity of voltage-dependent delayed rectifier potassium channel (Kv)

The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (Ky) were examined in normal and passively sensitized human airway smooth muscle (HASM), by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential (E m) were also detected. The results showed that phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group (P<0.05), and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the E m became more positive, as compared with the DMSO (a PMA menstruum)-treated group (P<0.01). This effect could be blocked by Ro31-8220 (P<0.01). It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.

XRCC1 Arg194Trp polymorphism and risk of chronic obstructive pulmonary disease.

The DNA damage, caused by cigarette smoking, can cause airway cell apoptosis and death, which may be associated with the development of chronic obstructive pulmonary disease (COPD). However, just 20%–30% smokers develop COPD, which suggests that different degrees of DNA repair cause different outcomes in smokers. X-ray repair cross-complementing group 1 (XRCC1), a base excision repair protein, has multiple roles in repairing ROS-mediated, basal DNA damage and single-strand DNA breaks. The present study investigated the association between polymorphism in XRCC1 (Arg399Gln) and susceptibility of COPD. A total of 201 COPD cases and 309 controls were recruited and frequency-matched on age and sex. XRCC1 genotype was determined by PCR-restriction fragment length polymorphism analysis. Overall, compared with those with the XRCC1 Arg/Arg genotype, the risk for COPD had no significant difference among individuals with Trp/Trp genotype. However, after stratifying by smoking status, in former smokers, compared with those with the XRCC1 Arg/Arg genotype, the risk for COPD was significantly reduced among individuals with Trp/Trp genotype (adjusted OR=0.22, 95% CI 0.06–0.85, P=0.028); after stratifying by smoking exposure, in light smokers, compared with those with the XRCC1 Arg/Arg genotype, the risk for COPD was significantly reduced among individuals with Arg/Trp genotype and Trp/Trp genotype (adjusted OR=0.39, 95% CI 0.16–0.94, P=0.036; 0.24, 95% CI 0.07–0.79, P=0.019, respectively). In conclusion, XRCC1 Arg194Trp genotype is associated with a reduced risk of developing COPD among former and light smokers.SummaryThe DNA damage, caused by cigarette smoking, can cause airway cell apoptosis and death, which may be associated with the development of chronic obstructive pulmonary disease (COPD). However, just 20%–30% smokers develop COPD, which suggests that different degrees of DNA repair cause different outcomes in smokers. X-ray repair cross-complementing group 1 (XRCC1), a base excision repair protein, has multiple roles in repairing ROS-mediated, basal DNA damage and single-strand DNA breaks. The present study investigated the association between polymorphism in XRCC1 (Arg399Gln) and susceptibility of COPD. A total of 201 COPD cases and 309 controls were recruited and frequency-matched on age and sex. XRCC1 genotype was determined by PCR-restriction fragment length polymorphism analysis. Overall, compared with those with the XRCC1 Arg/Arg genotype, the risk for COPD had no significant difference among individuals with Trp/Trp genotype. However, after stratifying by smoking status, in former smokers, compared with those with the XRCC1 Arg/Arg genotype, the risk for COPD was significantly reduced among individuals with Trp/Trp genotype (adjusted OR=0.22, 95% CI 0.06–0.85, P=0.028); after stratifying by smoking exposure, in light smokers, compared with those with the XRCC1 Arg/Arg genotype, the risk for COPD was significantly reduced among individuals with Arg/Trp genotype and Trp/Trp genotype (adjusted OR=0.39, 95% CI 0.16–0.94, P=0.036; 0.24, 95% CI 0.07–0.79, P=0.019, respectively). In conclusion, XRCC1 Arg194Trp genotype is associated with a reduced risk of developing COPD among former and light smokers.