cell biochemistry and function Cell Biochem Funct 2014; 32: 580–589. Published online 17 September 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3054
The functional study of human umbilical cord mesenchymal stem cells harbouring angiotensin-converting enzyme 2 in rat acute lung ischemia-reperfusion injury model Fabing Liu1†, Fengying Gao2†, Qian Li3 and Zhenwei Liu4* 1
Department of Department of 3 Department of 4 Department of 2
Thoracic Surgery, First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China Respiratory Medicine, Shanghai Jian Gong Hospital, Shanghai, China Pediatrics, First People’s Hospital of Kunshan, Jiangsu University, Kunshan, China Respiratory Medicine, First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China
Acute lung ischemia-reperfusion injury (ALIRI) is featured as non-specific alveolar damage, lung edema and hypoxemia, often occurring within 72 h after surgery. It is the leading cause for primary graft failure and mortality after lung surgery and transplantation. Here we aimed to find a more effective therapeutic approach to treat ALIRI. We evaluated the combinational effects of human umbilical cord mesenchymal stem cells (HUMSCs) and angiotensin-converting enzyme 2 (ACE2) in the rat ALIRI model. HUMSCs were isolated for lentiviral-ACE2 transfection. Fifty rats were randomly divided into five groups: sham surgery, physiological saline (PS), ACE2, HUMSCs and HUMSCs-ACE2 group. Several physiological, biochemical and histological indicators were examined and compared among the five groups, such as blood oxygen saturation (Sat O2%) and right ventricular systolic blood pressure (RVSBP), pulmonary morphology observations, several kinds of cell markers and the abundance of glutathione reductase (GR), glutathione peroxidase (GPX) and NAD(P)H quinone oxidoreductase (NQO1). Compared with HUMSCs and ACE2 groups, HUMSCs-ACE2 group showed lighter lung injuries, higher CD31 and vWF expression (endothelial cell surface markers), lower γ-H2AX (DNA damage marker) and CD68 (inflammatory cell marker) and higher anti-oxidants expression (GR, GPX and NQO1). The results indicated that HUMSCs harbouring ACE2 were more effective than either HUMSCs or ACE2 alone in alleviating the ALIRI damages. The synergistic effects of HUMSCs and ACE2 provide informative clues for mechanism study and therapeutic method development of ALIRI. Copyright © 2014 John Wiley & Sons, Ltd. key words—acute lung ischemia-reperfusion injury; human umbilical cord mesenchymal stem cells; angiotensin-converting enzyme 2; lung edema
INTRODUCTION Being characterized as refractory hypoxemia and respiratory distress, acute lung injury (ALI) is a systematic inflammatory response syndrome caused by kinds of intra- and extra-pulmonary pathogenic factors. As a common postoperative complication, acute respiratory distress syndrome has high incidence and mortality and is one of the leading causes for the death of patients in intense care unit.1–4 Acute lung ischemia-reperfusion injury (ALIRI) is the leading cause for ALI.1–3,5 ALIRI/ALI is a clinical pathological process, including lung transplantation, extracorporeal circulation, circulatory arrest and recovery, hypoxemic respiratory failure, smoke inhalation, pulmonary embolism, septicemia and so on.1–6 The current ALI therapeutic methods mainly focus on supportive treatments, such as protective ventilation
*Correspondence to: Zhenwei Liu, MD, PHD Department of Respiratory Medicine, First People’s Hospital, Affiliated to Shanghai Jiao Tong University, No. 100 Haining Road, Shanghai 200080, China. E-mail: [email protected] † Co-first authors: Fabing Liu and Fengying Gao.
Copyright © 2014 John Wiley & Sons, Ltd.
with slight incidence and mortality decreases.1,2 More effective ALI treatment methods are badly needed. Currently speaking, regardless of advances in unveiling interactions and expressional regulations of related inflammatory cells, cytokines and cell adhesion molecules, the underlying pathogenic mechanism is still unknown. Previous studies have shown that reactive oxygen species (ROS) generated from ALIRI/ALI inflammatory responses could deteriorate progressive lesions by damaging pulmonary parenchyma, gas exchange system and lung macrocirculation and microcirculation, revealing a great potential to develop new therapeutic methods based on preventing the production and amplification of inflammation, ROS and oxidative stress.7–9 Derived from mesoderm of early phase, mesenchymal stem cells (MSCs) are pluripotent stem cells with a great capacity for self-renewal while maintaining their multipotency. Recently, growing results have shown that MSCs are involved in inflammation suppression, antioxidative stress and anti-apoptotic process critical for lung tissue repair and regeneration.10–12 Because of the easy availability, low rate of virus contamination (the presence Received 29 April 2014 Revised 28 July 2014 Accepted 30 July 2014
581
humscs harbouring ace2 relieve aliri of fetal blood shelter) and zero ethical barrier, cord blood is the best resource for MSCs. Human umbilical cord mesenchymal stem cells (HUMSCs) are the research hotspot for pulmonary diseases, such as lung injuries and pulmonary congestion.13,14 MSCs could be used in curing pulmonary diseases by serving as a vector-carrying critical gene. Recent reports showed that MSCs and haematopoietic stem cells carrying keratinocyte growth factor are effective in curing pulmonary fibrosis.15 As a member of renin-angiotensin system, angiotensin-converting enzyme 2 (ACE2) is discovered in 2000.16 ACE2 is a specific exopeptidase expressed predominately in endothelial cells and could degrade some proinflammatory peptides, such as angiotensin II (Ang II), Ang IV and des-Arg9-BK.17–19 ACE2 catalyses Ang II into Ang (1-7), which inhibits smooth muscle cell proliferation and diastolizes the blood vessels.20 Ever since the discovery of being a critical receptor of severe acute respiratory syndrome virus in 2003, the protective roles of ACE2 in acute and chronic lung diseases have been revealed,21,22 such as antiinflammatory, anti-oxidation and anti-apoptosis.23 Thus, ACE2 has a great potential to be utilized in gene therapy.23 So far there is no report on combined effects of HUMSCs and ACE2 in ALIRI treatment. By using immunofluorescence and immunohistochemistry techniques, we investigated the protective roles of HUMSCs carrying ACE2 in rat ALIRI model. Our results provide informative evidences for gene and stem cell therapy development in ALIRI treatment.
MATERIALS AND METHODS Umbilical cord and animals The umbilical cords of healthy full term caesarean birth fetus were provided by the First People’s Hospital affiliated to Shanghai Jiao Tong University. All the experiments involving umbilical cords were conducted upon the agreement of pregnant women and approved by local ethics committee. Rattus norregiucs (No. 50; SD; female; 8 weeks; weight 250 ± 20 g) were kindly provided by the Department of Laboratory Animal Science, School of Medicine, Shanghai Jiao Tong University. Rats were divided in ten cages and fed with regular feed in well-ventilated room (free drinking; temperature: 21 ± 1 °C; 60% humidity;
The functional study of human umbilical cord mesenchymal stem cells harbouring angiotensin-converting enzyme 2 in rat acute lung ischemia-reperfusion injury model Fabing Liu1†, Fengying Gao2†, Qian Li3 and Zhenwei Liu4* 1
Department of Department of 3 Department of 4 Department of 2
Thoracic Surgery, First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China Respiratory Medicine, Shanghai Jian Gong Hospital, Shanghai, China Pediatrics, First People’s Hospital of Kunshan, Jiangsu University, Kunshan, China Respiratory Medicine, First People’s Hospital, Affiliated to Shanghai Jiao Tong University, Shanghai, China
Acute lung ischemia-reperfusion injury (ALIRI) is featured as non-specific alveolar damage, lung edema and hypoxemia, often occurring within 72 h after surgery. It is the leading cause for primary graft failure and mortality after lung surgery and transplantation. Here we aimed to find a more effective therapeutic approach to treat ALIRI. We evaluated the combinational effects of human umbilical cord mesenchymal stem cells (HUMSCs) and angiotensin-converting enzyme 2 (ACE2) in the rat ALIRI model. HUMSCs were isolated for lentiviral-ACE2 transfection. Fifty rats were randomly divided into five groups: sham surgery, physiological saline (PS), ACE2, HUMSCs and HUMSCs-ACE2 group. Several physiological, biochemical and histological indicators were examined and compared among the five groups, such as blood oxygen saturation (Sat O2%) and right ventricular systolic blood pressure (RVSBP), pulmonary morphology observations, several kinds of cell markers and the abundance of glutathione reductase (GR), glutathione peroxidase (GPX) and NAD(P)H quinone oxidoreductase (NQO1). Compared with HUMSCs and ACE2 groups, HUMSCs-ACE2 group showed lighter lung injuries, higher CD31 and vWF expression (endothelial cell surface markers), lower γ-H2AX (DNA damage marker) and CD68 (inflammatory cell marker) and higher anti-oxidants expression (GR, GPX and NQO1). The results indicated that HUMSCs harbouring ACE2 were more effective than either HUMSCs or ACE2 alone in alleviating the ALIRI damages. The synergistic effects of HUMSCs and ACE2 provide informative clues for mechanism study and therapeutic method development of ALIRI. Copyright © 2014 John Wiley & Sons, Ltd. key words—acute lung ischemia-reperfusion injury; human umbilical cord mesenchymal stem cells; angiotensin-converting enzyme 2; lung edema
INTRODUCTION Being characterized as refractory hypoxemia and respiratory distress, acute lung injury (ALI) is a systematic inflammatory response syndrome caused by kinds of intra- and extra-pulmonary pathogenic factors. As a common postoperative complication, acute respiratory distress syndrome has high incidence and mortality and is one of the leading causes for the death of patients in intense care unit.1–4 Acute lung ischemia-reperfusion injury (ALIRI) is the leading cause for ALI.1–3,5 ALIRI/ALI is a clinical pathological process, including lung transplantation, extracorporeal circulation, circulatory arrest and recovery, hypoxemic respiratory failure, smoke inhalation, pulmonary embolism, septicemia and so on.1–6 The current ALI therapeutic methods mainly focus on supportive treatments, such as protective ventilation
*Correspondence to: Zhenwei Liu, MD, PHD Department of Respiratory Medicine, First People’s Hospital, Affiliated to Shanghai Jiao Tong University, No. 100 Haining Road, Shanghai 200080, China. E-mail: [email protected] † Co-first authors: Fabing Liu and Fengying Gao.
Copyright © 2014 John Wiley & Sons, Ltd.
with slight incidence and mortality decreases.1,2 More effective ALI treatment methods are badly needed. Currently speaking, regardless of advances in unveiling interactions and expressional regulations of related inflammatory cells, cytokines and cell adhesion molecules, the underlying pathogenic mechanism is still unknown. Previous studies have shown that reactive oxygen species (ROS) generated from ALIRI/ALI inflammatory responses could deteriorate progressive lesions by damaging pulmonary parenchyma, gas exchange system and lung macrocirculation and microcirculation, revealing a great potential to develop new therapeutic methods based on preventing the production and amplification of inflammation, ROS and oxidative stress.7–9 Derived from mesoderm of early phase, mesenchymal stem cells (MSCs) are pluripotent stem cells with a great capacity for self-renewal while maintaining their multipotency. Recently, growing results have shown that MSCs are involved in inflammation suppression, antioxidative stress and anti-apoptotic process critical for lung tissue repair and regeneration.10–12 Because of the easy availability, low rate of virus contamination (the presence Received 29 April 2014 Revised 28 July 2014 Accepted 30 July 2014
581
humscs harbouring ace2 relieve aliri of fetal blood shelter) and zero ethical barrier, cord blood is the best resource for MSCs. Human umbilical cord mesenchymal stem cells (HUMSCs) are the research hotspot for pulmonary diseases, such as lung injuries and pulmonary congestion.13,14 MSCs could be used in curing pulmonary diseases by serving as a vector-carrying critical gene. Recent reports showed that MSCs and haematopoietic stem cells carrying keratinocyte growth factor are effective in curing pulmonary fibrosis.15 As a member of renin-angiotensin system, angiotensin-converting enzyme 2 (ACE2) is discovered in 2000.16 ACE2 is a specific exopeptidase expressed predominately in endothelial cells and could degrade some proinflammatory peptides, such as angiotensin II (Ang II), Ang IV and des-Arg9-BK.17–19 ACE2 catalyses Ang II into Ang (1-7), which inhibits smooth muscle cell proliferation and diastolizes the blood vessels.20 Ever since the discovery of being a critical receptor of severe acute respiratory syndrome virus in 2003, the protective roles of ACE2 in acute and chronic lung diseases have been revealed,21,22 such as antiinflammatory, anti-oxidation and anti-apoptosis.23 Thus, ACE2 has a great potential to be utilized in gene therapy.23 So far there is no report on combined effects of HUMSCs and ACE2 in ALIRI treatment. By using immunofluorescence and immunohistochemistry techniques, we investigated the protective roles of HUMSCs carrying ACE2 in rat ALIRI model. Our results provide informative evidences for gene and stem cell therapy development in ALIRI treatment.
MATERIALS AND METHODS Umbilical cord and animals The umbilical cords of healthy full term caesarean birth fetus were provided by the First People’s Hospital affiliated to Shanghai Jiao Tong University. All the experiments involving umbilical cords were conducted upon the agreement of pregnant women and approved by local ethics committee. Rattus norregiucs (No. 50; SD; female; 8 weeks; weight 250 ± 20 g) were kindly provided by the Department of Laboratory Animal Science, School of Medicine, Shanghai Jiao Tong University. Rats were divided in ten cages and fed with regular feed in well-ventilated room (free drinking; temperature: 21 ± 1 °C; 60% humidity;
