Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions.MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry.ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14⁺ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells.ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.     

Thiazolidinedione inhibits the production of monocyte chemoattractant protein-1 in cytokine-treated human vascular endothelial cells.

The chemokine monocyte chemoattractant protein-1 is a potent chemoattractant for monocytes. Monocyte chemoattractant protein-1 is produced by vascular endothelial cells during inflammatory diseases such as atherosclerosis. In this study, we examined the effects of a thiazolidinedione on monocyte chemoattractant protein-1 expression in human vascular endothelial cells. In human vascular endothelial cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous monocyte chemoattractant protein-1 protein secretion, mRNA expression and promoter activity. The thiazolidinedione inhibited these effects. In summary, our results indicated that the suppression of the expression of monocyte chemoattractant protein-1 can be accomplished by thiazolidinedione treatment, raising the possibility that thiazolidinedione may be of therapeutic value in the treatment of diseases such as atherosclerosis.     

Bone marrow-derived mesenchymal stromal cells support rat pancreatic islet survival and insulin secretory function in vitro

Background aimsRecent evidence has suggested that transplanted bone marrow (BM)-derived mesenchymal stromal cells (MSC) are able to engraft and repair non-hematopoietic tissues successfully, including central nervous system, renal, pulmonary and skin tissue, and may possibly contribute to tissue regeneration. We examined the cytoprotective effect of BM MSC on co-cultured, isolated pancreatic isletsMethodsPancreatic islets and MSC isolated from Lewis rats were divided into four experimental groups: (a) islets cultured alone (islet control); (b) islets cultured in direct contact with MSC (IM-C); (c) islets co-cultured with MSC in a Transwell system, which allows indirect cell contact through diffusible media components (IM-I); and (d) MSC cultured alone (MSC control). The survival and function of islets were measured morphologically and by analyzing insulin secretion in response to glucose challenge. Cytokine profiles were determined using a cytokine array and enzyme-linked immunosorbent assaysResultsIslets contact-cultured with MSC (IM-C) showed sustained survival and retention of glucose-induced insulin secretory function. In addition, the levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were decreased, and tissue inhibitor of metalloproteinases-1 (TIMP-1) and vascular endothelial growth factor (VEGF) levels were increased at 4 weeks in both the IM-C and IM-I groupsConclusionsThese results indicate that contact co-culture is a major factor that contributes to islet survival, maintenance of cell morphology and insulin function. There might also be a synergic effect resulting from the regulation of inflammatory cytokine production. We propose that BM MSC are suitable for generating a microenvironment favorable for the repair and longevity of pancreatic islets.     

Isogenic mesenchymal stem cells transplantation improves a rat model of chronic aristolochic acid nephropathy via upregulation of hepatic growth factor and downregulation of transforming growth factor β1

Chronic aristolochic acid (AA) nephropathy (CAAN) caused by intake of AA-containing herbs is difficult to treat. We evaluated the therapeutic effect of bone marrow (BM) mesenchymal stem cells (MSCs) on a rat model of CAAN. Female Wistar rats were fed with decoction of Caulis Aristolochia manshuriensis by intragastric administration. MSCs were prepared from BM of male Wistar rats and injected into female CAAN rats through tail vein. Body weight, renal function, and urinary excretion of these CAAN rats were monitored before killing at the end of the 20th week. Blood, urine, and tissue samples were collected from experimental (MSC and non-MSC) and normal control groups. All animals developed renal fibrosis after 12 weeks of intake of AA-containing decoction. Fibrosis in the MSC groups was significantly reduced as examined with light and electron microscopy. Blood urea nitrogen, serum creatinine, and urine protein levels were significantly reduced and hemoglobin levels were improved in the MSC group as compared with the non-MSC group (p < 0.01). The expression of TGF-β1 mRNA and protein was reduced but hepatic growth factor (HGF) was increased in the MSC group compared with the non-MSC group, but still higher than the normal control level as measured by immunochemical, RT-PCR, and western blotting assays (p < 0.01). The renal fibrosis of CAAN could be protected by isogenic MSC transplantation, probably via upregulation of HGF and downregulation of TGF-β1.     

Role of mesenchymal stem cells on cornea wound healing induced by acute alkali burn

The aim of this study was to investigate the effects of subconjunctivally administered mesenchymal stem cells (MSCs) on corneal wound healing in the acute stage of an alkali burn. A corneal alkali burn model was generated by placing a piece of 3-mm diameter filter paper soaked in NaOH on the right eye of 48 Sprague-Dawley female rats. 24 rats were administered a subconjunctival injection of a suspension of 2×10(6) MSCs in 0.1 ml phosphate-buffered saline (PBS) on day 0 and day 3 after the corneal alkali burn. The other 24 rats were administered a subconjunctival injection of an equal amount of PBS as a control. Deficiencies of the corneal epithelium and the area of corneal neovascularization (CNV) were evaluated on days 3 and 7 after the corneal alkali burn. Infiltrated CD68(+) cells were detected by immunofluorescence staining. The mRNA expression levels of macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were analyzed using real-time polymerase chain reaction (real-time PCR). In addition, VEGF protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). MSCs significantly enhanced the recovery of the corneal epithelium and decreased the CNV area compared with the control group. On day 7, the quantity of infiltrated CD68(+) cells was significantly lower in the MSC group and the mRNA levels of MIP-1α, TNF-α, and VEGF and the protein levels of VEGF were also down-regulated. However, the expression of MCP-1 was not different between the two groups. Our results suggest that subconjunctival injection of MSCs significantly accelerates corneal wound healing, attenuates inflammation and reduces CNV in alkaline-burned corneas; these effects were found to be related to a reduction of infiltrated CD68(+) cells and the down-regulation of MIP-1α, TNF-α and VEGF.     

Mesenchymal stromal cell injection protects against oxidative stress in Escherichia coli–induced acute lung injury in mice

Background aimsStem cells may be a promising therapy for acute respiratory distress syndrome. Recent in vivo and in vitro studies suggested that the mesenchymal stromal cells (MSCs) have anti-oxidative stress properties. We hypothesized that intravenous injection of bone marrow–derived mesenchymal stem cells (MSCs) could attenuate Escherichia coli–induced acute lung injury (ALI) in mice by controlling the oxidative stress status.MethodsEighty mice were randomly divided into four groups: group 1 (control group) received 25 μL of saline as a vehicle; group 2 contained E coli–induced ALI mice; group 3 included mice that received MSCs before induction of ALI; group 4 included mice that received MSCs after induction of ALI. Lung samples were isolated and assayed for oxidative stress variables and histopathologic analysis. Total anti-oxidant capacity was measured in broncho-alveolar lavage.ResultsPre- and post-injury MSC injection increased survival, reduced pulmonary edema and attenuated lung injuries in ALI mice. Histologically, MSCs exhibited a considerable degree of preservation of the pulmonary alveolar architecture. An increase of anti-oxidant enzyme activities and a decrease of myeloperoxidase activity and malondialdehyde levels in the MSC recipient groups versus the ALI group were found. Furthermore, the total anti-oxidant capacity and reduced glutathione levels were significantly increased in MSCs recipient groups versus the ALI group. Weak +ve inducible nitric oxide synthase immuno-expression in groups that received MSCs was detected. Pre-injury MSC injection showed better effects than did post-injury MSC injection.ConclusionsSystemic bone marrow–derived MSC injection was effective in modulating the oxidative stress status in E coli–induced acute lung injury in mice.     

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.     

Mechanistic action of mesenchymal stem cell injection in the treatment of chemically induced ovarian failure in rabbits

BackgroundNo curative treatment is known for primary ovarian failure; however, mesenchymal stem cells (MSCs), through self-renewal and regeneration, push the trial to evaluate their role in the treatment of ovarian failure. The aim of this study was to explore the impact of MSCs on cyclophosphamide (CTX)-induced ovarian failure in rabbits and to clarify the mechanism(s) by which MSCs exert their action.MethodsThirty-five adult female rabbits were injected with CTX to induce ovarian failure. Five rabbits were euthanized after the last injection of CTX for histological examination. The others (30 rabbits) were further subdivided into two groups: group 1 (ovarian failure group, 15 rabbits) received no treatment; group 2 (ovarian failure and MSC recipient group, 15 rabbits) received MSCs isolated from extracted bone marrow of male rabbits.ResultsA decrease of follicle-stimulating hormone and an increase of estrogen and vascular endothelial growth factor (VEGF) levels in the MSC recipient group versus the ovarian failure group were found. Weak caspase-3 expression and +ve proliferating cell nuclear antigen staining after MSC injection were detected. Cytological and histological examinations showed increased follicle numbers with apparent normal structure of ovarian follicles in the MSC recipient group. Moreover, Y chromosome–containing cells from male donors were present within the ovarian tissues in group 2.ConclusionsThe current study suggests that intravenous injection of MSCs into rabbits with chemotherapy-induced ovarian damage improved ovarian function. MSCs accomplish this function by direct differentiation into specific cellular phenotypes and by secretion of VEGF, which influence the regeneration of the ovary.     

利妥昔单抗治疗肾病综合征患者效果观察及对视黄醇结合蛋白、微循环状态的影响分析

摘要 目的：探讨与分析利妥昔单抗（RTX）治疗肾病综合征患者效果观察及对视黄醇结合蛋白（RBP）、微循环状态的影响。方法：2018年9月到2021年11月选择在本院诊治的肾病综合征患者66例作为研究对象,所有患者按入院先后顺序编号,依据治疗方法分为利妥昔单抗组和对照组各33例,对照组给予糖皮质激素治疗,利妥昔单抗组在对照组治疗的基础上给予利妥昔单抗治疗,治疗观察3个月,观察与检测血清RBP、微循环状态变化情况。结果：治疗后利妥昔单抗组的总有效率为97.0 %,明显高于对照组的81.8 %（P<0.05）。两组治疗后的尿蛋白定量、尿蛋白定性水平低于治疗前,利妥昔单抗组明显低于对照组,而血浆白蛋白较治疗前高,且利妥昔单抗组高于对照组（P<0.05）。两组治疗后的甲襞微循环管绊形态、流态评分明显低于治疗前,利妥昔单抗组明显低于对照组（P<0.05）。两组治疗后的血清RBP含量低于治疗前,利妥昔单抗组明显低于对照组（P<0.05）。两组治疗期间不良反应对比无明显差异（P>0.05）。结论：利妥昔单抗治疗肾病综合征患者能有效改善微循环状态,抑制血清RBP的表达,能提高治疗效果,还可促进改善肾功能,具有安全性。     

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice

Background aimsCo-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation.MethodsMouse islets and kidney MSCs were co-encapsulated in alginate, and their function was assessed in vitro. A minimal mass of 350 syngeneic islets encapsulated alone or co-encapsulated with MSCs (islet+MSC) were transplanted intraperitoneally into diabetic mice, and blood glucose concentrations were monitored. Capsules were recovered 6 weeks after transplantation, and islet function was assessed.ResultsIslets co-encapsulated with MSCs in vitro had increased glucose-stimulated insulin secretion and content. The average blood glucose concentration of transplanted mice was significantly lower by 3 weeks in the islet+MSC group. By week 6, 71% of the co-encapsulated group were cured compared with 16% of the islet-alone group. Capsules recovered at 6 weeks had greater glucose-stimulated insulin secretion and insulin content in the islet+MSC group.ConclusionsMSCs improved the efficacy of microencapsulated islet transplantation. Using an isolated-graft model, we were able to eliminate the impact of MSC-mediated enhancement of revascularization and preservation of islet morphology and demonstrate that the improvement in insulin secretion and content is sustained in vivo and can significantly improve graft outcome.     

Mesenchymal stem cell sheet transplantation combined with locally released simvastatin enhances bone formation in a rat tibia osteotomy model

Nonunion of fractured bones is a common clinical problem for orthopedic surgeons. This study aimed to investigate the effects of simvastatin locally applied from calcium sulfate (CS) combined with a mesenchymal stem cell (MSC) sheet on fracture healing. In vitro, the proliferation and differentiation of rat bone marrow–derived MSCs stimulated by simvastatin were investigated. In vivo, an osteotomy model was made in rat tibia, and fractured tibias were treated with CS, CS/simvastatin, CS/MSC sheet or simvastatin-loaded CS with MSC or untreated (control). Tibias were harvested at 2 or 8 weeks and underwent real-time quantitative polymerase chain reaction, x-ray, micro-CT and histological analysis. The expression levels of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor of simvastatin-induced MSCs increased with the concentrations of the simvastatin, significantly higher than those in the MSCs group. At 2 weeks, the CS/simvastatin/MSC sheet group showed significantly higher expressions of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor, with more callus formation around the fracture site compared with the other four groups. At 8 weeks, complete bone union was obtained in the CS/simvastatin/MSC sheet group. By contrast, newly regenerated bone tissue partially bridged the gap in the CS/simvastatin group and the CS/MSC sheet group; the control and CS group showed nonunion of the tibia. These results show that both simvastatin and the MSC sheet contributed to the formation of new bone and that the tibia fracture was completely healed by transplantation of the MSC sheet with locally applied simvastatin. Such MSC sheet with locally applied simvastatin might contribute to the treatment of fractures, bone delayed unions or nonunions in clinical practice.     

Mesenchymal stem cells modified with angiopoietin-1 improve remodeling in a rat model of acute myocardial infarction

We used human angiopoietin-1 (hAng1)-modified mesenchymal stem cells (MSCs) to treat acute myocardial infarction (AMI) in rats. The hAng1 gene was transfected into cultured rat MSCs using an adenoviral vector. Five million hAng-transfected MSCs (MSC(Ang1)) or green fluorescent protein transfected MSCs (MSC(GFP)) or PBS only (PBS group) were injected intramyocardially into the inbred Lewis rat hearts immediately after myocardial infarction. MSC(Ang1) survived in the infarcted myocardium, and expressed hAng1 at both mRNA and protein levels. The vascular density was higher in the MSC(Ang1) and MSC(GFP) groups than in the PBS group. The measurements of infarcted ventricular wall thickness, infarction area, and left ventricular diameter indicated that heart remodeling was inhibited and heart function was improved in both the MSC(Ang1) and MSC(GFP) groups. However, in contrast to the MSC(GFP) group, the MSC(Ang1) group showed enhanced angiogenesis and arteriogenesis (by 11-35%), infarction area was reduced by 30% and the left ventricular wall was 46% thicker (P<0.05). The results indicated that hAng1-modified MSCs improved heart function, followed by angiogenic effects in salvaging ischemic myocardium and reduced cardiac remodeling.     

Chemokine amplification in mesangial cells.

Mesangial cells are specialized cells of the renal glomerulus that share some properties of vascular smooth muscle cells and macrophages. They are implicated in the pathogenesis of many forms of nephritis. The murine CXC-chemokines macrophage inflammatory protein-2 (MIP-2) and KC induce migration of mouse mesangial cells. Mesangial cells also exhibit a unique chemokine feedback mechanism. Treatment with nanomolar concentrations of MIP-2 or KC markedly up-regulates monocyte chemoattractant protein-1 and RANTES expression in mesangial cells. Autoinduction of MIP-2 and KC mRNA was also noted. Low levels of MIP-1alpha, MIP-1beta, and IFN-gamma-inducible protein-10 were induced following treatment with higher doses of MIP-2 or KC. These effects are specific to mesangial cells, as MIP-2 or KC treatment of renal cortical epithelial cells or peritoneal macrophages failed to induce chemokine production. This cascade of chemokine interactions may contribute to renal infiltration and leukocyte activation. The abilities of MIP-2 or KC to stimulate their own synthesis may also contribute to the maintenance and chronic course of glomerular inflammation. The mesangial cell receptor for MIP-2 and/or KC is unknown but is not CXC-chemokine receptor-2.     

Dietary restriction suppresses inflammation and delays the onset of stroke in stroke-prone spontaneously hypertensive rats

Subjects with high blood levels of inflammatory markers and patients with chronic inflammatory disorders are at high risk for stroke. Dietary restriction (DR) suppresses systemic inflammation to deter age-related chronic diseases. To examine whether DR delays the onset of stroke, 10-week-old stroke-prone spontaneously hypertensive rats (SHRSP) were assigned to either a control (ad libitum) or DR (50% diet of control) group, and day of stroke onset and lifespan were observed. DR markedly delayed the onset of stroke in SHRSP compared to control without affecting blood pressure. Day of stroke onset (median) in the control group was 34 days, whereas it was 70 days in the DR group. After 2 weeks of DR and before the onset of stroke, plasma levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) and their mRNA expression levels in adipose tissue were significantly lower in the DR rats than in the control rats. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression levels in cerebrovascular endothelial cells (CVECs), and macrophage infiltration into brain were lower in the DR rats than in the control rats. IL-1β and TNF-α treatment in CVECs increased MCP-1, C-reactive protein, ICAM-1, and VCAM-1 mRNA and their protein levels in vitro. In conclusion, suppression of inflammation in response to DR may lead to a delay in the onset of stroke independent of any effect on blood pressure in SHRSP.     

Lignin-derived lignophenols attenuate oxidative and inflammatory damage to the kidney in streptozotocin-induced diabetic rats

Abstract

This study investigated the effects of lignin-derived lignophenols (LPs) on the oxidative stress and infiltration of macrophages in the kidney of streptozotocin (STZ)-induced diabetic rats. The diabetic rats were divided into four groups with 0%, 0.11%, 0.33% and 1.0% LP diets. The vehicle-injected controls were given a commercial diet. At 5 weeks, superoxide (O₂^?) production, macrophage kinetics, the degree of fibrosis in glomeruli and mRNA expression for monocyte chemoattractant protein-1 (MCP-1) were examined. The NADPH-stimulated O₂^? levels in the kidney of the diabetic rats treated with 1.0% LP were significantly lower than those in untreated diabetic rats. The number of macrophages, levels of MCP-1 mRNA expression and degree of glomerular fibrosis increased in untreated LP and these levels were significantly lower in 1.0%LP-treated rats. The results suggested that LPs suppress the excess oxidative stress, the infiltration and activation of macrophages and the glomerular expansion in STZ-induced diabetic kidneys.     

Effect of bone marrow–derived mesenchymal stromal cells on hepatoma

Background aimsThe aim of the study was to evaluate the effect of mesenchymal stromal cells (MSCs) on tumor cell growth in vitro and in vivo and to elucidate the apoptotic and anti-proliferative mechanisms of MSCs on a hepatocellular carcinoma (HCC) murine model.MethodsThe growth-inhibitory effect of MSCs on the Hepa 1–6 cell line was tested by means of methyl thiazolyl diphenyl-tetrazolium assay. Eighty female mice were randomized into four groups: group 1 consisted of 20 mice that received MSCs only by intrahepatic injection; group 2 consisted of 20 HCC mice induced by inoculation of Hepa 1–6 cells into livers without MSC treatment; group 3 consisted of 20 mice that received MSCs after induction of liver cancer; group 4 consisted of 20 mice that received MSCs after induction of liver cancer on top of induced biliary cirrhosis.ResultsMSCs exhibited a growth-inhibitory effect on Hepa 1–6 murine cell line in vitro. Concerning in vivo study, decreases of serum alanine transaminase, aspartate transaminase and albumin levels after MSC transplantation in groups 2 and 3 were found. Gene expression of α-fetoprotein was significantly downregulated after MSC injection in the HCC groups. We found that gene expression of caspase 3, P21 and P53 was significantly upregulated, whereas gene expression of Bcl-2 and survivin was downregulated in the HCC groups after MSC injection. Liver specimens of the HCC groups confirmed the presence of dysplasia. The histopathological picture was improved after administration of MSCs to groups 2 and 3.ConclusionsMSCs upregulated genes that help apoptosis and downregulated genes that reduce apoptosis. Therefore, MSCs could inhibit cell division of HCC and potentiate their death.