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Background Experimental and clinical trials have demonstrated the efficiency of bone marrow-derived mesenchymal stromal/stem cells (bMSCs) in the treatment of myocardial infarction. However, after intravenous injection, the ineffective migration of engrafted bMSCs to the hearts remains an obstacle, which has an undesirable impact on the efficiency of cell-based therapy. Therefore, we attempted to identify a marker that could distinguish a subpopulation of bMSCs with a promising migratory capacity. Methods Here, CD51-negative and CD51-positive cells were isolated by flow cytometry from Ter119−CD45−CD31−bMSCs and cultured in specifically modified medium. The proliferation ability of the cells was evaluated by 5-ethynyl-2′-deoxyuridine (EdU) staining or continuously monitored during culture, and the differentiation potential was assessed by culturing the cells in the appropriate conditioned media. Wound healing assays, transwell assays and quantitative polymerase chain reaction (qPCR) were used to measure the migratory ability. The mice were subjected to a sham operation or myocardial infarction (MI) by permanently occluding the coronary artery, and green fluorescent protein (GFP)-labelled cells were transplanted into the mice via intravenous infusion immediately after MI. Heart function was measured by echocardiography; infarct myocardium tissues were detected by triphenyl tetrazolium chloride (TTC) staining. Additionally, immunofluorescence staining was used to verify the characteristics of CD51+bMSCs and inflammatory responses in vivo. Statistical comparisons were performed using a two-tailed Student’s t test. Results In this study, the isolated CD51−bMSCs and CD51+bMSCs, especially the CD51+ cells, presented a favourable proliferative capacity and could differentiate into adipocytes, osteocytes and chondrocytes in vitro. After the cells were transplanted into the MI mice by intravenous injection, the therapeutic efficiency of CD51+bMSCs in improving left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) was better than that of CD51−bMSCs. Compared with CD51−bMSCs, CD51+bMSCs preferentially migrated to and were retained in the infarcted hearts at 48 h and 8 days after intravenous injection. Accordingly, the migratory capacity of CD51+bMSCs exceeded that of CD51−bMSCs in vitro, and the former cells expressed higher levels of chemokine receptors or ligands. Interestingly, the retained CD51+bMSCs retained in the myocardium possessed proliferative potential but only differentiated into endothelial cells, smooth muscle cells, fibroblasts or cardiomyocytes. Transplantation of CD51+bMSCs partially attenuated the inflammatory response in the hearts after MI, while the potential for inflammatory suppression was low in CD51−bMSC-treated mice. Conclusions These findings indicated that the CD51-distinguished subpopulation of bMSCs facilitated proliferation and migration both in vitro and in vivo, which provided a novel cell-based strategy to treat acute MI in mice by intravenous injection.

A high-fat diet is a major risk factor for coronary heart diseases. Matrix metalloprotease (MMP) expression is changed in many cardiovascular diseases. Selenium, which is an important trace element in animals, has a close relationship with cardiovascular diseases. The TGFβ1/Smad signalling pathway is ubiquitous in diverse tissues and cells, and it is also associated with the occurrence and development of cardiovascular diseases. Therefore, in this study, we aimed to determine selenium's effect on lipid metabolism, atherosclerotic plaque formation, and MMP2 expression, as well as the underlying functional mechanism.In vivo tests: 24 male New Zealand white rabbits were randomly divided into 4 groups: regular diet, high-fat diet, high-fat diet+selenium and regular diet+selenium groups. The high-fat diet induced the lipid disturbances of rabbits at week 12. Selenium supplementation lowered total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels (p0.01). Selenium supplementation also suppressed MMP2 over-expression in thoracic aortas. In vitro tests: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of selenium or ox-LDL. Ox-LDL promoted MMP2 expression by increasing TGFβ1, pSmad2, pSmad3 and Smad3 expression (p0.01). Selenium attenuated MMP2 over-expression by regulating the TGFβ1/Smad signalling pathway.Selenium suppressed high-fat diet-induced MMP2 over-expression in vivo by improving lipid metabolism. In vitro, selenium attenuated MMP2 over-expression through the TGFβ1/Smad signalling pathway.

Our previous study demonstrated that angiotensin II (Ang II) upregulates the expression of Kv1.5, a promising target for atrial fibrillation (AF) therapy, by activating ROS-dependent P-Smad2/3 and P-ERK 1/2. A recent study showed that hydrogen sulfide (H2S) may modulate the effects of angiotensin II (Ang II) by inhibiting the NADPH oxidase 4 (Nox4)-ROS signaling in the heart. The present study aimed to determine whether H2S is involved in the regulation of atrial Kv1.5 via ROS-related mechanisms in AF. Cultured neonatal rat atrial myocytes and a beagle model of AF were used for this study. In the neonatal rat atrial myocytes, quantitative PCR and enzyme immunoassays revealed that the mRNA expression levels of angiotensinogen, angiotensin-converting enzyme, and Ang II type I receptor (AT1R) and the Ang II supernatant concentration were significantly increased by hydrogen peroxide (H2O2) incubation, and these H2O2-induced alterations were reversed by diphenyleneiodonium, apocynin and H2S supplementation. Flow cytometry and Western blotting revealed that blockade of H2S biosynthesis using dl-propargylglycine increased ROS production and the expression of Ang II and Kv1.5. Sodium hydrosulfide (an exogenous H2S donor) and Nox4 siRNA inhibited Ang II-induced ROS production and Ang II-induced expression of Kv1.5, P-Smad2/3, P-ERK 1/2. Sodium hydrosulfide suppressed the Ang II-induced upregulation of Nox4. In our beagle AF model, 24 h of rapid atrial pacing (RAP) increased the atrial Ang II concentration, ROS production and the protein expression of Nox4, Kv1.5, P-Smad2/3 and P-ERK 1/2. These RAP-induced changes were inhibited by H2S supplementation and losartan (an AT1R blocker) pretreatment. In conclusion, our study indicates that H2S downregulates Ang II-induced atrial Kv1.5 expression by attenuating Nox4-related ROS-triggered P-Smad2/3 and P-ERK 1/2 activation during AF. H2S supplementation would be beneficial for AF treatment via the suppression of atrial Kv1.5 expression.

Our previous investigation indicated that angiotensin II (Ang II) enhances the expression of Kv1.5, a promising target for the treatment of atrial fibrillation (AF), by activating reactive oxygen species (ROS)-dependent phosphorylation of Smad 2/3 (forming P-Smad 2/3) and ERK 1/2 (forming P-ERK 1/2). A recent study indicated that aldosterone (Aldo) upregulates atrial Kv1.5 protein in a rat AF model, but the mechanism remains unknown. The present study aimed to clarify the mechanism underlying Aldo-induced Kv1.5 expression and to test whether spironolactone may modulate atrial Kv1.5. Our Western blot analysis indicated that the Aldo/mineralocorticoid receptor (MR) interacts with Ang II/AT1R in upregulating Kv1.5 expression in cultured neonatal atrial myocytes (NRAMs). Blockade of MR with spironolactone and of AT1R with losartan significantly suppressed Kv1.5 expression induction by combined Aldo and Ang II treatment. Aldo increased the protein expression of Nox1, Nox2 and Nox4, but this effect was abolished by spironolactone pretreatment. The Aldo-induced upregulation of Kv1.5 was also reversed by the Src protein tyrosine kinase family inhibitor PP2, the Nox2 inhibitor gp91ds-tat and the Nox1/Nox4 inhibitor GKT137831 but not by the Rac GTPase inhibitor NSC23766. Flow cytometry showed that the Aldo-induced ROS production was inhibited by spironolactone, PP2, gp91ds-tat and GKT137831. Spironolactone suppressed the Aldo-induced protein expression phosphorylated Src (P-Src), P-Smad 2/3 and P-ERK 1/2. In conclusion, we have demonstrated that spironolactone suppresses Aldo-induced Kv1.5 expression by attenuating MR-Nox1/2/4-mediated ROS generation in NRAMs.

Additional file 1. Supplementary methods and figures. (DOCX 1275 kb)

Although neutrophils play critical roles in innate immunity, in excess these cells cause severe tissue damage. Thus, neutrophil activation must be tightly regulated to prevent indiscriminant damage. Previously, we reported that mice lacking matrix metalloproteinase (MMP) 7 are protected from lung injury owing to markedly impaired neutrophil movement from the interstitium into mucosal lumenal spaces. This phenotype resulted from a lack of MMP7 shedding of syndecan-1, a heparan sulfate proteoglycan that carries the neutrophil chemokine CXCL1 as cargo. Here, we assessed if shedding syndecan-1/CXCL1 complexes affects neutrophil activation. Whereas injured monolayers of wild-type alveolar type II cells potently stimulated neutrophil activation, as gauged by release of myeloperoxidase, cells from Mmp7(-/-) or syndecan-1-null (Sdc1(-/-)) mice or human cells with MMP7 knockdown did not. In vivo, we observed reduced myeloperoxidase release relative to neutrophil numbers in bleomycin-injured Mmp7(-/-) and Sdc1(-/-) mice. Furthermore, we determined that soluble syndecan-1 directly stimulated neutrophil activation in the absence of cellular damage. These data indicate that MMP7 shedding of syndecan-1/CXCL1 complexes functions as a checkpoint that restricts neutrophil activation at sites of epithelial injury.

Objective The aim of this study is to investigate local cardiac and circulating AVP secretion during heart failure and to determine whether AVP mediates ventricular remodeling. Methods We assessed cardiac function and AVP levels of post-myocardial infarction (MI) heart-failure rats 3 weeks (n = 10), 4 weeks (n = 10), 6 weeks (n = 10), 9 weeks (n = 15) after the proximal left anterior descending coronary artery (LAD) ligation. Ten sham-operated rats were used as the control group. In vitro, cardiac microvascular endothelial cells (CMECs) were initiated from isolated Wistar rat hearts and subjected to Ang II to induce AVP expression and secretion. Besides, the effects of AVP stimulation on CMECs and cardiac fibroblasts (CFs) were studied using methylthiazol tetrazolium assay, Western blotting and real-time PCR. Results With cardiac dysfunction, plasma and local cardiac AVP, aldosterone levels increased over time, peaking at 9 weeks post-MI. AVP levels were negatively correlated with serum Na + and LVEF but positively correlated with LVEDD and myocardial hydroxyproline. In CMECs treated with Ang II, AVP mRNA and protein expression increased. In addition, AVP promoted CFs proliferation and up-regulated the expression of endothelin-1 and connective tissue growth factor. Conclusion CMECs are the cellular sources of elevated local heart AVP stimulated with Ang II/AT1. An intrinsic cardiac AVP system exists. Local cardiac and circulating AVP secretion were enhanced by deteriorating cardiac function. AVP may promote ventricular remodeling. Thus, AVP could be an important mediator of myocardial fibrosis in late-stage heart failure.

Lipid disturbance induced by high-fat diet is a worldwide problem, and it can induce inflammation and oxidative stress in vivo. Zinc is considered as an antioxidant, anti-inflammatory agent. Since matrix metalloprotease 2 (MMP2) and matrix metalloprotease 9 (MMP9)'s expressions are changed under many pathological conditions, we would like to know how zinc affects lipid metabolism and MMP2, MMP9's expressions in the lipid disturbance rabbits. Twenty-four male New Zealand white rabbits were randomly divided into four groups. Each group had six rabbits, and they were fed with regular diet, high-fat diet, high-fat diet+zinc, and regular diet+zinc separately for 12 weeks. High-fat diet induced lipid disturbance significantly which raised the level of aspartate aminotransferase (p0.01) and alanine transaminase (p0.05) in the high-fat diet group, but zinc supplement reversed this phenomenon (p0.05). Zinc did not reduce total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (p0.05), but it lowered triglyceride (TG) and raised high-density lipoprotein cholesterol (HDL-C) (p0.01). Zinc also reduced high-sensitivity C-reactive protein (hs-CRP) (p0.01) and interleukin-6 (IL-6)'s expressions (p0.05). Zinc reduced the epicardial adipose tissue and alleviated the hepatic steatosis. Zinc suppressed MMP2 and MMP9's expressions in vivo, but it did not alleviate the aorta fatty streak's severity in the lipid disturbance rabbits. Zinc protected the liver, reduced TG, hs-CRP, and IL-6 and raised HDL-C in the lipid disturbance rabbits. Zinc suppressed MMP2 and MMP9's expressions in vivo, but it did not alleviate the severity of aorta fatty streak induced by the high-fat diet.

Matrilysin (matrix metalloproteinase-7) is highly expressed in lungs of patients with pulmonary fibrosis and other conditions associated with airway and alveolar injury. Although matrilysin is required for closure of epithelial wounds ex vivo, the mechanism of its action in repair is unknown. We demonstrate that matrilysin mediates shedding of E-cadherin ectodomain from injured lung epithelium both in vitro and in vivo. In alveolar-like epithelial cells, transfection of activated matrilysin resulted in shedding of E-cadherin and accelerated cell migration. In vivo, matrilysin co-localized with E-cadherin at the basolateral surfaces of migrating tracheal epithelium, and the reorganization of cell-cell junctions seen in wild-type injured tissue was absent in matrilysin-null samples. E-cadherin ectodomain was shed into the bronchoalveolar lavage fluid of bleomycin-injured wild-type mice, but was not shed in matrilysin-null mice. These findings identify E-cadherin as a novel substrate for matrilysin and indicate that shedding of E-cadherin ectodomain is required for epithelial repair.

Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7-28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.

Epithelial-mesenchymal interactions are of critical importance during tissue morphogenesis and repair. Although the cellular and molecular aspects of many of these interactions are beginning to be understood, the ability of epithelial cells to regulate fibroblast interstitial matrix production has not been extensively studied. We report here that cultured alveolar epithelial cells are capable of modulating the expression of tropoelastin, the soluble precursor of the interstitial lung matrix component elastin, by lung fibroblasts. Phorbol ester-stimulated alveolar epithelial cells secrete a soluble factor that causes a time- and dose-dependent repression of lung fibroblast tropoelastin mRNA expression. This alveolar epithelial cell-mediated repressive activity is specific for tropoelastin, is effective on lung fibroblasts from multiple stages of development, and acts at the level of transcription. Partial characterization of the repressive activity indicates it is an acid-stable, pepsin-labile protein. Gel fractionation of alveolar epithelial cell conditioned medium revealed two peaks of activity with relative molecular masses of ∼25 and 50 kDa. These data support a role for epithelial cells in the regulation of fibroblast interstitial matrix production.

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen. M. leprae can infect a variety of cells in vivo, including epithelial cells, muscle cells, and Schwann cells, in addition to macrophages. The ligand-receptor interactions important in the attachment and ingestion of M. leprae by these nonmacrophage cells remains unknown. Fibronectin (FN) significantly enhances both attachment and ingestion of M. leprae by epithelial and Schwann cell lines. We cloned an M. leprae FN binding protein (FN attachment protein [FAP]) distinct from the 85ABC complex which has been shown previously to bind FN. The FAP open reading frame predicts a protein of 29.5 kDa with a 39-amino-acid signal peptide and was previously described as an antigen in leprosy patients. M. leprae FAP has homologies in M. vaccae, M. avium, and M. tuberculosis, as determined by Southern blotting and direct peptide analysis. Both anti-FAP antibodies and an Escherichia coli-expressed recombinant protein significantly blocked M. leprae attachment and internalization by T-24, an epithelial cell line, and JS1, a Schwann cell line. These data suggest that FN can be a bridging opsonic ligand for attachment of mycobacteria to nonphagocytes and that FAP plays an important role in this process. This may be an important step in the initiation of M. leprae infection in vivo.

BackgroundLung injury promotes the expression of matrix metalloproteinase-7 (MMP7, matrilysin), which is required for neutrophil recruitment and re-epithelialization. MMP7 governs the lung inflammatory response through the shedding of syndecan-1. Because inflammation and repair are related events, we evaluated the role of syndecan-1 shedding in lung re-epithelialization.Methodology/principal findingEpithelial injury induced syndecan-1 shedding from wild-type epithelium but not from Mmp7(-/-) mice in vitro and in vivo. Moreover, cell migration and wound closure was enhanced by MMP7 shedding of syndecan-1. Additionally, we found that syndecan-1 augmented cell adhesion to collagen by controlling the affinity state of the alpha(2)beta(1) integrin.Conclusion/significanceMMP7 shedding of syndecan-1 facilitates wound closure by causing the alpha(2)beta(1) integrin to assume a less active conformation thereby removing restrictions to migration. MMP7 acts in the lungs to regulate inflammation and repair, and our data now show that both these functions are controlled through the shedding of syndecan-1.

The influx of inflammatory cells to sites of injury is largely directed by signals from the epithelium, but how these cells form chemotactic gradients is not known. In matrilysin null mice, neutrophils remained confined in the interstitium of injured lungs and did not advance into the alveolar space. Impaired transepithelial migration was accompanied by a lack of both shed syndecan-1, a heparan sulfate proteoglycan, and KC, a CXC chemokine, in the alveolar fluid. KC was bound to shed syndecan-1, and it was not detected in the lavage of syndecan-1 null mice. In vitro, matrilysin cleaved syndecan-1 from the surface of cells. Thus, matrilysin-mediated shedding of syndecan-1/KC complexes from the mucosal surface directs and confines neutrophil influx to sites of injury.