Qi-Hong Chen

Interaction between mesenchymal stem cells and endothelial cells restores endothelial permeability via paracrine hepatocyte growth factor in vitro

IntroductionMesenchymal stem cells (MSCs) have potent stabilising effects on vascular endothelium injury, inhibiting endothelial permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secrete more factors by MSC-endothelial cell (MSC-EC) interactions. We hypothesised that MSC-EC interactions restore endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF.MethodsWe investigated the endothelial permeability induced by LPS under two co-culture conditions. Human pulmonary microvascular endothelial cells (HPMECs) were added into the upper chambers of cell-culture inserts, while two different co-culture conditions were used in the lower side of the transwells, as follows: (1) MSC-EC interaction group: MSCs and HPMECs contact co-culture; (2) MSC group: MSCs only. The endothelial paracellular and transcellular permeabilities in the upper side of transwells were detected. Then the concentration of HGF was measured in the culture medium by using an enzyme-linked immunosorbent assay kit, followed by neutralisation of HGF with anti-HGF antibody in the co-culture medium. In addition, adherens junction and cytoskeleton protein expressions were measured by Western blot and immunofluorescence. HPMEC proliferation was analysed by bromodeoxyuridine incorporation assay.ResultsThe paracellular permeability significantly increased after LPS stimulation in a dose-dependent and time-dependent manner. Meanwhile, MSC-EC interaction more significantly decreased endothelial paracellular and transcellular permeability induced by LPS. Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group. However, neutralising HGF with anti-HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability. Compared with the MSC group, MSC-EC interaction increased vascular endothelial (VE)-cadherin and occludin protein expression, reduced caveolin-1 protein expression in HPMECs, and restored remodelling of F-actin and junctional localisation of VE-cadherin. Furthermore, the proliferation ratio in the MSC-EC interaction group was higher than that of the MSC group. However, the effects of MSCs were significantly blocked by anti-HGF antibody.ConclusionsThese data suggested that MSC-EC interaction decreased endothelial permeability induced by LPS, which was attributed mainly to HGF secreted by MSCs. The main mechanisms by which HGF restored the integrity of endothelial monolayers were remodelling of endothelial intercellular junctions, decreasing caveolin-1 protein expression, and inducing proliferation in HPMECs.

Wnt5a through Noncanonical Wnt/JNK or Wnt/PKC Signaling Contributes to the Differentiation of Mesenchymal Stem Cells into Type II Alveolar Epithelial Cells In Vitro

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells is critical for reepithelization and recovery in acute respiratory distress syndrome (ARDS), and Wnt signaling was considered to be the underlying mechanisms. In our previous study, we found that canonical Wnt pathway promoted the differentiation of MSCs into AT II cells, however the role of the noncanonical Wnt pathway in this process is unclear. It was disclosed in this study that noncanonical Wnt signaling in mouse bone marrow–derived MSCs (mMSCs) was activated during the differentiation of mMSCs into AT II cells in a modified co-culture system with murine lung epithelial-12 cells and small airway growth media. The levels of surfactant protein (SP) C, SPB and SPD, the specific markers of AT II cells, increased in mMSCs when Wnt5a was added to activate noncanonical Wnt signaling, while pretreatment with JNK or PKC inhibitors reversed the promotion of Wnt5a. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. We found that the Wnt5a supplement promoted the vertical and horizontal migration of mMSCs, ameliorated the cell death and the reduction of Bcl-2/Bax induced by H2O2. The effect of Wnt5a on the migration of mMSCs and their survival after H2O2 exposure were partially inhibited with PKC or JNK blockers. In conclusion, Wnt5a through Wnt/JNK signaling alone or both Wnt/JNK and Wnt/PKC signaling promoted the differentiation of mMSCs into AT II cells and the migration of mMSCs; through Wnt/PKC signaling, Wnt5a increased the survival of mMSCs after H2O2 exposure in vitro.

Activation of Wnt/β-catenin signalling promotes mesenchymal stem cells to repair injured alveolar epithelium induced by lipopolysaccharide in mice

IntroductionMesenchymal stem cells (MSCs) have potential for re-epithelization and recovery in acute respiratory distress syndrome (ARDS). In a previous in vitro study, the results showed that the canonical Wnt/β-catenin pathway promoted the differentiation of MSCs into type II alveolar epithelial cells, conferred resistance to oxidative stress, and promoted their migration, suggesting that the Wnt/β-catenin pathway might be one of the key mechanisms underling the therapeutic effect of mouse MSCs in ARDS.MethodsMouse MSCs stable transfected with β-catenin or green fluorescent protein control were transplanted intratracheally into the ARDS mice induced by lipopolysaccharide. Lung tissue injury and repair assessment were examined using haematoxylin and eosin staining, lung injury scoring, Masson’s trichrome staining and fibrosis scoring. Homing and differentiation of mouse MSCs were assayed by labelling and tracing MSCs using NIR815 dye, immunofluorescent staining, and Western immunoblot analysis. The inflammation and permeability were evaluated by detecting the cytokine and protein measurements in bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay.ResultsIn this study, β-catenin-overexpressing MSC engraftment led to more significant effects than the GFP controls, including the retention of the MSCs in the lung, differentiation into type II alveolar epithelial cells, improvement in alveolar epithelial permeability, and the pathologic impairment of the lung tissue.ConclusionThese results suggest that the activation of canonical Wnt/β-catenin pathway by mouse MSCs by overexpressing β-catenin could further improve the protection of mouse MSCs against epithelial impair and the therapeutic effects of mouse MSCs in ARDS mice.

Mesenchymal Stem Cells Overexpressing Angiotensin-Converting Enzyme 2 Rescue Lipopolysaccharide-Induced Lung Injury.

Bone marrow-derived mesenchymal stem cells (MSCs), which have beneficial effects in acute lung injury (ALI), can serve as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2), a counterregulatory enzyme of ACE that degrades angiotensin (Ang) II into Ang 1–7, has a protective role against ALI. Because ACE2 expression is severely reduced in the injured lung, a therapy targeted to improve ACE2 expression in lung might attenuate ALI. We hypothesized that MSCs overexpressing ACE2 would have further benefits in lipopolysaccharide (LPS)-induced ALI mice, when compared with MSCs alone. MSCs were transduced with ACE2 gene (MSC-ACE2) by a lentiviral vector and then infused into wild-type (WT) and ACE2 knockout (ACE2-/y) mice following an LPS-induced intratracheal lung injury. The results demonstrated that the lung injury of ALI mice was alleviated at 24 and 72 h after MSC-ACE2 transplantation. MSC-ACE2 improved the lung histopathology and had additional anti-inflammatory effects when compared with MSCs alone in both WT and ACE2-/y ALI mice. MSC-ACE2 administration also reduced pulmonary vascular permeability, improved endothelial barrier integrity, and normalized lung eNOS expression relative to the MSC group. The beneficial effects of MSC-ACE2 could be attributed to its recruitment into the injured lung and enhanced local expression of ACE2 protein without changing the serum ACE2 levels after MSC-ACE2 transplantation. The biological activity of the increased ACE2 protein decreased the Ang II amount and increased the Ang 1–7 level in the lung when compared with the ALI and MSC-only groups, thereby inhibiting the detrimental effects of accumulating Ang II. Therefore, compared to MSCs alone, the administration of MSCs overexpressing ACE2 resulted in a further improvement in the inflammatory response and pulmonary endothelial function of LPS-induced ALI mice. These additional benefits could be due to the degradation of Ang II that accompanies the targeted overexpression of ACE2 in the lung.

Stable Genetic Alterations of β-Catenin and ROR2 Regulate the Wnt Pathway, Affect the Fate of MSCs

The Wnt pathways have been shown to be critical for the fate of mesenchymal stem cells (MSCs) in vitro, but their roles in MSCs in vivo remain poorly characterized due to the lack of stable alterations in their signaling. In the present study, we constructed long‐term and stable mMSCs lines with activated and inactivated β‐catenin (the key molecule of the canonical Wnt signaling pathway) or ROR2 (the key molecule of the noncanonical Wnt5a/ROR2 signaling pathway) modifications with lentiviral vectors. We found that the transduction efficiencies mediated by the lentiviral vectors were 92.61–97.04% and were maintained over 20 passages of mMSCs. Transfection by lentiviral vectors not only regulated the mRNA and protein expression of β‐catenin or ROR2 but also regulated nuclear β‐catenin accumulation or the Wnt5a/JNK and Wnt5a/PKC pathways belonging to the canonical Wnt and noncanonical Wnt5a/ROR2 pathways, respectively. β‐Catenin or ROR2 gene overexpression promoted mMSC proliferation, migration and differentiation into osteoblasts, while inhibiting the adipogenic differentiation of mMSCs. In contrast, inactivation of the β‐catenin or ROR2 genes resulted in the opposite effects. Therefore, these results confirm that lentiviral vector transduction can facilitate sustained and efficient gene modification of the Wnt pathway in mMSCs. This study provides a method to investigate the effects of the Wnt pathway on the fate of mMSCs in vivo and for the further improvement of MSC‐based therapies. J. Cell. Physiol. 229: 791–800, 2014.

Comparison of the effects of albumin and crystalloid on mortality in adult patients with severe sepsis and septic shock: a meta-analysis of randomized clinical trials

IntroductionThe aim of this study was to examine whether albumin reduced mortality when employed for the resuscitation of adult patients with severe sepsis and septic shock compared with crystalloid by meta-analysis.MethodsWe searched for and gathered data from MEDLINE, Elsevier, Cochrane Central Register of Controlled Trials and Web of Science databases. Studies were eligible if they compared the effects of albumin versus crystalloid therapy on mortality in adult patients with severe sepsis and septic shock. Two reviewers extracted data independently. Disagreements were resolved by discussion with other two reviewers until a consensus was achieved. Data including mortality, sample size of the patients with severe sepsis, sample size of the patients with septic shock and resuscitation endpoints were extracted. Data were analyzed by the methods recommended by the Cochrane Collaboration Review Manager 4.2 software.ResultsA total of 5,534 records were identified through the initial search. Five studies compared albumin with crystalloid. In total, 3,658 severe sepsis and 2,180 septic shock patients were included in the meta-analysis. The heterogeneity was determined to be non-significant (P = 0.86, I2 = 0%). Compared with crystalloid, a trend toward reduced 90-day mortality was observed in severe sepsis patients resuscitated with albumin (odds ratio (OR) 0.88; 95% CI, 0.76 to 1.01; P = 0.08). However, the use of albumin for resuscitation significantly decreased 90-day mortality in septic shock patients (OR 0.81; 95% CI, 0.67 to 0.97; P = 0.03). Compared with saline, the use of albumin for resuscitation slightly improved outcome in severe sepsis patients (OR 0.81; 95% CI, 0.64 to 1.08; P = 0.09).ConclusionsIn this meta-analysis, a trend toward reduced 90-day mortality was observed in severe sepsis patients resuscitated with albumin compared with crystalloid and saline. Moreover, the 90-day mortality of patients with septic shock decreased significantly.

The Effect of Early Goal-Directed Therapy on Outcome in Adult Severe Sepsis and Septic Shock Patients: A Meta-Analysis of Randomized Clinical Trials.

BACKGROUND:Whether early goal-directed therapy (EGDT) improves outcome in severe sepsis and septic shock remains unclear. We performed a meta-analysis of existing clinical trials to examine whether EGDT improved outcome in the resuscitation of adult sepsis patients compared with control care. METHODS:We searched for eligible studies using MEDLINE, Elsevier, Cochrane Central Register of Controlled Trials, and Web of Science databases. Studies were eligible if they compared the effects of EGDT versus control care on mortality in adult patients with severe sepsis and septic shock. Two reviewers extracted data independently. Data including mortality, sample size of the patients with severe sepsis and septic shock, and resuscitation end points were extracted. Data were analyzed using methods recommended by the Cochrane Collaboration Review Manager 4.2 software. Random errors were evaluated by trial sequential analysis (TSA). RESULTS:Nine studies compared EGDT with control care, and 5202 severe sepsis and septic shock patients were included. A nonsignificant trend toward reduction in the longest all-cause mortality was observed in the EGDT group compared with control care (relative risk, 0.89; 99% confidence interval, 0.74–1.07; P = 0.10). However, EGDT significantly reduced intensive care unit mortality in severe sepsis and septic shock patients (relative risk, 0.72; 99% confidence interval, 0.57–0.90; P = 0.0002). TSA indicated lack of firm evidence for a beneficial effect. CONCLUSIONS:In this meta-analysis, a nonsignificant trend toward reduction in the longest all-cause mortality in patients resuscitated with EGDT was noted. However, EGDT significantly reduced intensive care unit mortality in severe sepsis and septic shock patients. TSA indicated a lack of firm evidence for the results. More powered, randomized controlled trials are needed to determine the effects.

MSCs modified with ACE2 restore endothelial function following LPS challenge by inhibiting the activation of RAS.

Angiotensin (Ang) II plays an important role in the process of endothelial dysfunction in acute lung injury (ALI) and is degraded by angiotensin‐converting enzyme2 (ACE2). However, treatments that target ACE2 to injured endothelium and promote endothelial repair of ALI are lacking. Mesenchymal stem cells (MSCs) are capable of homing to the injured site and delivering a protective gene. Our study aimed to evaluate the effects of genetically modified MSCs, which overexpress the ACE2 protein in a sustained manner via a lentiviral vector, on Ang II production in endothelium and in vitro repair of lipopolysaccharide (LPS)‐induced endothelial injury. We found that the efficiency of lentiviral vector transduction of MSCs was as high as 97.8% and was well maintained over 30 passages. MSCs modified with ACE2 showed a sustained high expression of ACE2 mRNA and protein. The modified MSCs secreted soluble ACE2 protein into the culture medium, which reduced the concentration of Ang II and increased the production of Ang 1–7. MSCs modified with ACE2 were more effective at restoring endothelial function than were unmodified MSCs, as shown by the enhanced survival of endothelial cells; the downregulated production of inflammatory mediators, including ICAM‐1, VCAM‐1, TNF‐α, and IL‐6; reduced paracellular permeability; and increased expression of VE‐cadherin. These data demonstrate that MSCs modified to overexpress the ACE2 gene can produce biologically active ACE2 protein over a sustained period of time and have an enhanced ability to promote endothelial repair after LPS challenge. These results encourage further testing of the beneficial effects of ACE2‐modified MSCs in an ALI animal model. J. Cell. Physiol. 230: 691–701, 2015.

Therapeutic Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Models of Pulmonary and Extrapulmonary Acute Lung Injury.

Bone marrow-derived mesenchymal stem cells (MSCs) offer a promising therapy for acute lung injury (ALI). However, whether the same MSC treatments possess similar potential for different ALI models is not fully clear. The present study evaluated the distribution and therapeutic effects of intravenous MSC administration for the treatment of intratracheal lipopolysaccharide (LPS)-induced intrapulmonary ALI and intravenous LPS/zymosan-induced extrapulmonary ALI, matched with lung injury severity, at 30 min and 1, 3, and 7 days. We found that MSC transplantation attenuated lung injury and inhibited lung inflammation in both ALI models. The benefits of MSCs were more significant in the intrapulmonary ALI mice. In vivo and ex vivo fluorescence imaging showed that MSCs primarily homed into the lung. However, more MSCs were recruited into the lungs of the intrapulmonary ALI mice than those of the extrapulmonary ALI mice over the time course. A few MSCs were also detected in the liver and spleen at days 3 and 7. In addition, the two ALI models showed different extrapulmonary organ dysfunction. A lower percentage of cell apoptosis and SDF-1α levels was found in the liver and spleen of the intrapulmonary ALI mice than in those of the extrapulmonary ALI mice. These results suggested that the two ALI models were accompanied with different degrees of extrapulmonary organ damage, which resulted in differences in the trafficking and accumulation of MSCs to the injured lung and consequently accounted for different therapeutic effects of MSCs for lung repair in the two ALI models. These data suggest that intravenous administration of MSCs has a greater potential for the treatment of intrapulmonary ALI than extrapulmonary ALI matched with lung injury severity; these differences were due to more recruitment of MSCs in the lungs of intrapulmonary ALI mice than those of extrapulmonary ALI mice. This finding may contribute to the clinical use of MSCs for the treatment of ALI.

The Orphan Receptor Tyrosine Kinase ROR2 Facilitates MSCs to Repair Lung Injury in ARDS Animal Model

There are some limitations to the therapeutic effects of mesenchymal stem cells (MSCs) on acute respiratory distress syndrome (ARDS) due to their low engraftment and differentiation rates in lungs. We found previously that noncanonical Wnt5a signaling promoted the differentiation of mouse MSCs (mMSCs) into type II alveolar epithelial cells (AT II cells), conferred resistance to oxidative stress, and promoted migration of MSCs in vitro. As receptor tyrosine kinase-like orphan receptor 2 (ROR2) is an essential receptor for Wnt5a, it was reasonable to deduce that ROR2 might be one of the key molecules for the therapeutic effect of MSCs in ARDS. The mMSCs that stably overexpressed ROR2 or the green fluorescent protein (GFP) control were transplanted intratracheally into the ARDS mice [induced by intratracheal injection of lipopolysaccharide (LPS)]. The results showed that ROR2-overexpressing mMSCs led to more significant effects than the GFP controls, including the retention of the mMSCs in the lung, differentiation into AT II cells, improvement of alveolar epithelial permeability, improvement of acute LPS-induced pulmonary inflammation, and, finally, reduction of the pathological impairment of the lung tissue. In conclusion, MSCs that overexpress ROR2 could further improve MSC-mediated protection against epithelial impairment in ARDS.