3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney

Three‐dimensional (3D) cell culture has been reported to increase the therapeutic potentials of mesenchymal stem cells (MSCs). In this study, we aimed to investigate the therapeutic effects of 3D spheroids of human adipose‐derived MSCs for acute kidney injury (AKI). In vitro studies indicated that 3D spheroids of MSCs produced higher levels of extracellular matrix proteins (including collagen I, fibronectin and laminin), and exhibited stronger anti‐apoptotic and anti‐oxidative capacities than two‐dimensional (2D) cultured cells. Furthermore, 3D culture increased the paracrine secretion of cytokines by MSCs, including angiogenic factors (VEGF and basic fibroblast growth factor), anti‐apoptotic factors (epidermal growth factor and hepatocyte growth factor), the anti‐oxidative factor insulin‐like growth factor and the anti‐inflammatory protein tumour necrosis factor‐alpha stimulated gene/protein 6. Consistent with in vitro experiments, 3D spheroids of MSCs showed enhanced survival and paracrine effects in vivo. More importantly, when injected into the kidney of model rats with ischemia‐reperfusion (I/R)‐induced AKI, 3D spheroids were more beneficial in protecting the I/R kidney against apoptosis, reducing tissue damage, promoting vascularization and ameliorating renal function compared with 2D cultured cells. Therefore, the 3D culture strategy improved the therapeutic effects of MSCs, and might be promising for AKI treatment.     

Antithrombin III prevents progression of chronic kidney disease following experimental ischaemic‐reperfusion injury

Acute kidney disease (AKI) leads to increased risk of progression to chronic kidney disease (CKD). Antithrombin III (ATIII) is a potent anticoagulant with anti‐inflammatory properties, and we previously reported that insufficiencies of ATIII exacerbated renal ischaemia‐reperfusion injury (IRI) in rats. In this study, we examined the characteristic of AKI‐CKD transition in rats with two distinct AKI models. Based on our observation, left IRI plus right nephrectomy (NX‐IRI) was used to determine whether ATIII had therapeutic effects in preventing CKD progression after AKI. It was observed that NX‐IRI resulted in significant functional and histological damage at 5 weeks after NX‐IRI compared with sham rats, which was mitigated by ATIII administration. Besides, we noticed that ATIII administration significantly reduced NX‐IRI‐induced interstitial fibrosis. Consistently, renal expression of collagen‐1, α‐smooth muscle actin and fibronectin were substantial diminished in ATIII‐administered rats compared with un‐treated NX‐IRI rats. Furthermore, the beneficial effects of ATIII were accompanied with decreased M1‐like macrophage recruitment and down‐regulation of M1‐like macrophage‐dependent pro‐inflammatory cytokines such as tumour necrosis factor α, inducible nitric oxide synthase and interleukin‐1β, indicating that ATIII prevented AKI‐CKD transition via inhibiting inflammation. Overall, ATIII shows potential as a therapeutic strategy for the prevention of CKD progression after AKI.     

Renalase contributes to the renal protection of delayed ischaemic preconditioning via the regulation of hypoxia‐inducible factor‐1α

Ischaemic preconditioning (IPC) attenuates acute kidney injury (AKI) from renal ischaemia reperfusion. Renalase, an amine oxidase secreted by the proximal tubule, not only degrades circulating catecholamines but also protects against renal ischaemia reperfusion injury. Here, it has been suggested that the renoprotective effect of renal IPC is partly mediated by renalase. In a model of brief intermittent renal IPC, the increased cortex renalase expression was found to last for 48 hrs. IPC significantly reduced renal tubular inflammation, necrosis and oxidative stress following renal ischaemia reperfusion injury. Such effects were attenuated by blocking renalase with an anti‐renalase monoclonal antibody. We further demonstrated that renalase expression was up‐regulated by hypoxia in vitro via an hypoxia‐inducible factor (HIF)‐1α mechanism. The IPC‐induced up‐regulation of renalase in vivo was also reduced by pre‐treatment with an HIF‐1α inhibitor, 3‐(5′‐Hydroxymethyl‐2′‐furyl)‐1‐benzyl indazole. In summary, the renoprotective effect of IPC is partly dependent on the renalase expression, which may be triggered by hypoxia via an HIF‐1α mechanism. Endogenous renalase shows potential as a therapeutic agent for the prevention and treatment of AKI.     

Subcutaneous Construction of Engineered Adipose Tissue with Fat Lobule-Like Structure Using Injectable Poly-Benzyl-L-Glutamate Microspheres Loaded with Adipose-Derived Stem Cells

Porous microcarriers were fabricated from synthesized poly(γ-benzyl-L-glutamate) (PBLG) polymer to engineer adipose tissue with lobule-like structure via the injectable approach. The adipogenic differentiation of human adipose-derived stem cells (hASCs) seeded on porous PBLG microcarriers was determined by adipogenic gene expression and glycerol-3-phosphate dehydrogenase enzyme activity. In vitro adipogenic cultivation was performed for 7 days, and induced hASC/PBLG complex (Adi-ASC/PBLG group) was subcutaneously injected into nude mice. Injections of PBLG microcarriers alone (PBLG group) and non-induced hASC/PBLG complex (ASC/PBLG group) served as controls. Newly formed tissues were harvested after 4 and 8 weeks. Generation of subcutaneous adipose tissue with typical lobule-like structure separated by fibrous septa was observed upon injection of adipogenic-induced hASC/microsphere complex. Adipogenesis significantly increased in the Adi-ASC/PBLG group compared with the control groups. The angiogenesis in the engineered adipose tissue was comparable to that in normal tissue as determined by capillary density and luminal diameter. Cell tracking assay demonstrated that labeled hASCs remained detectable in the neo-generated tissues 8 weeks post-injection using green fluorescence protein-labeled hASCs. These results indicate that adipose tissue with typical lobule-like structure could be engineered using injectable porous PBLG microspheres loaded with adipogenic-induced hASCs.     

Promotion of human adipose‐derived stem cell proliferation mediated by exogenous nucleosides

Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose‐derived stem cells), with or without co‐treatment with 5‐aza (5‐azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5‐aza induces a dose‐dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 μM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5‐Aza‐induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.     

Bone Marrow�Cderived Endothelial Progenitor Cells and Endothelial Cells May Contribute to Endothelial Repair in the Kidney Immediately After Ischemia�CReperfusion

In ischemic acute kidney injury, renal blood flow is decreased. We have previously shown that reperfused, transplanted kidneys exhibited ischemic injury to vascular endothelium and that preservation of peritubular capillary endothelial integrity may be critical to recovery from ischemic injury. We hypothesized that bone marrow–derived (BMD) endothelial progenitor cells (EPCs) might play an important role in renal functional recovery after ischemia. We tested this hypothesis in recipients of cadaveric renal allografts before and for 2 weeks after transplantation. We found that the numbers of circulating CD34-positive EPCs and CD146-positive endothelial cells (ECs) decreased immediately after ischemia–reperfusion. In renal allograft tissues obtained 1 hr after reperfusion, CD34-positive cells were more frequently observed along the endothelial lining of peritubular capillaries compared with non-ischemic controls. Moreover, 0–17.5% of peritubular capillary ECs were of recipient origin. In contrast, only 0.1–0.7% of tubule cells were of recipient origin. Repeat graft biopsy samples obtained 35 and 73 days after transplant did not contain capillary ECs of recipient origin, whereas 1.4% and 12.1% of tubule cells, respectively, were of recipient origin. These findings suggest that BMD EPCs and ECs may contribute to endothelial repair immediately after ischemia–reperfusion. (J Histochem Cytochem 58:687–694, 2010)     

Agent-Based Model of Therapeutic Adipose-Derived Stromal Cell Trafficking during Ischemia Predicts Ability To Roll on P-Selectin

Intravenous delivery of human adipose-derived stromal cells (hASCs) is a promising option for the treatment of ischemia. After delivery, hASCs that reside and persist in the injured extravascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limit the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic hASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting bottlenecks in the homing process. This process, however, is incredibly complex, which merited the use of computational techniques to speed the rate of discovery. We developed a multicell agent-based model (ABM) of hASC trafficking during acute skeletal muscle ischemia, based on over 150 literature-based rules instituted in Netlogo and MatLab software programs. In silico, trafficking phenomena within cell populations emerged as a result of the dynamic interactions between adhesion molecule expression, chemokine secretion, integrin affinity states, hemodynamics and microvascular network architectures. As verification, the model reasonably reproduced key aspects of ischemia and trafficking behavior including increases in wall shear stress, upregulation of key cellular adhesion molecules expressed on injured endothelium, increased secretion of inflammatory chemokines and cytokines, quantified levels of monocyte extravasation in selectin knockouts, and circulating monocyte rolling distances. Successful ABM verification prompted us to conduct a series of systematic knockouts in silico aimed at identifying the most critical parameters mediating hASC trafficking. Simulations predicted the necessity of an unknown selectin-binding molecule to achieve hASC extravasation, in addition to any rolling behavior mediated by hASC surface expression of CD15s, CD34, CD62e, CD62p, or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 µm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.     

Differentiation of human adipose‐derived stem cells into beating cardiomyocytes

Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca²⁺ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.     

Treatment with Actovegin improves spatial learning and memory in rats following transient forebrain ischaemia

This study aimed to investigate whether Actovegin, which is a deproteinized ultrafiltrate derived from calf blood, demonstrates neuroprotective effects in a rat model of transient global cerebral ischaemia. Forty Sprague Dawley rats were subjected to four‐vessel occlusion to induce transient global cerebral ischaemia followed by either saline or Actovegin treatment. Sham operations were performed on 15 rats. Actovegin (200 mg/kg) or saline was administered 6 hrs after carotid artery occlusion and then daily until Day 40. Learning and memory were evaluated using the Morris water maze test over two different 5‐day periods, and grip strength testing was also performed to control for potential motor impairments. Rat brains were harvested for histological analysis on Day 68. In comparison to controls, Actovegin‐treated rats exhibited a decreased latency to reach the hidden platform on the second learning trial of water maze testing (46.82 ± 6.18 versus 27.64 ± 4.53 sec., P < 0.05; 38.3 ± 8.23 versus 13.37 ± 2.73 sec., P < 0.01 for the first and second 5‐day testing periods, respectively). In addition, Actovegin‐treated rats spent more time in the platform quadrant than saline‐treated rats during memory trials (P < 0.05). No differences in grip strength were detected. Histological analyses demonstrated increased cell survival in the CA1 region of the hippocampus following Actovegin treatment (left hemisphere, 166 ± 50 versus 332 ± 27 cells, P < 0.05; right hemisphere, 170 ± 45 versus 307 ± 28 cells, P < 0.05, in saline‐ versus Actovegin‐treated rats, respectively). In rats, Actovegin treatment improves spatial learning and memory following cerebral ischaemia, which may be related to hippocampal CA1 neuroprotection.     

Adipogenic differentiation of human adipose-derived stem cells grown as spheroids

Understanding the process of adipogenesis is critical if suitable therapeutics for obesity and related metabolic diseases are to be found. The current study presents proof of feasibility of creating a 3-D spheroid model using human adipose-derived stem cells (hASCs) and their subsequent adipogenic differentiation. hASC spheroids were formed atop an elastin-like polypeptide-polyethyleneimine (ELP-PEI) surface and differentiated using an adipogenic cocktail. Spheroids were matured in the presence of dietary fatty acids (linoleic or oleic acid) and evaluated based on functional markers including intracellular protein, CD36 expression, triglyceride accumulation, and PPAR-γ gene expression. Spheroid size was found to increase as the hASCs matured in the adipocyte maintenance medium, though the fatty acid treatment generally resulted in smaller spheroids compared to control. A stable protein content over the 10-day maturation period indicated contact-inhibited proliferation as well as minimal loss of spheroids during culture. Spheroids treated with fatty acids showed greater amounts of intracellular triglyceride content and greater expression of the key adipogenic gene, PPAR-γ. We also demonstrated that 3-D spheroids outperformed 2-D monolayer cultures in adipogenesis. We then compared the adipogenesis of hASC spheroids to that in 3T3-L1 spheroids and found that the triglyceride accumulation was less profound in hASC spheroids than that in 3T3-L1 adipocytes, correlated with smaller average spheroids, suggesting a relatively slower differentiation process. Taken together, we have shown the feasibility of adipogenic differentiation of patient-derived hASC spheroids, which with further development, may help elucidate key features in the adipogenesis process.     

Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox‐sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin‐secreting Rin‐m5f β‐cells and its role in autocrine and paracrine MP‐mediated cell crosstalk under conditions of oxidative stress. MP from aPC‐treated primary endothelial (EC) or β‐cells were applied to H₂O₂‐treated Rin‐m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26‐stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR‐1 expression in EC and to a lesser extent in β‐cells. MP from aPC‐treated EC (eM_aPC) exhibited high EPCR and annexin A1 content, protected β‐cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1‐dependent mechanism. eMP activated EPCR/PAR‐1 and ANXA1/FPR2‐dependent pathways and up‐regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H₂O₂‐treated rat islets with increased viability (62% versus 48% H₂O₂), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.     

The perioperative time course and clinical significance of the chemokine CXCL16 in patients undergoing cardiac surgery

The chemokine CXCL16 and its receptor CXCR6 have been linked to the pathogenesis of acute and chronic cardiovascular disease. However, data on the clinical significance of CXCL16 in patients undergoing cardiac surgery with acute myocardial ischemia/reperfusion (I/R) are still lacking. Therefore, we determined CXCL16 in the serum of cardiac surgery patients and investigated its kinetics and association with the extent of organ dysfunction. 48 patients underwent conventional cardiac surgery with myocardial I/R and the use of cardiopulmonary bypass (CPB) were consecutively enrolled in the present study. We investigated the peri‐ and post‐operative profile of CXCL16. Clinical relevant data were assessed and documented throughout the entire observation period. To identify the influence of myocardial I/R and CPB on CXCL16 release data were compared to those received from patients that underwent off‐pump procedure. Pre‐operative serum CXCL16 levels were comparable to those obtained from healthy volunteers (1174 ± 55.64 pg/ml versus 1225 ± 70.94). However, CXCL16 levels significantly increased during surgery (1174 ± 55.64 versus 1442 ± 75.42 pg/ml; P = 0.0057) and reached maximum levels 6 hrs after termination of surgery (1174 ± 55.64 versus 1648 ± 74.71 pg/ml; P < 0.001). We revealed a positive correlation between the intraoperative serum levels of CXCL16 and the extent of organ dysfunction (r² = 0.356; P = 0.031). Patients with high CXCL16 release showed an increased extent of organ dysfunction compared to patients with low CXCL16 release. Our study shows that CXCL16 is released into the circulation as a result of cardiac surgery and that high post‐operative CXCL16 levels are associated with an increased severity of post‐operative organ dysfunctions.     

Deficiency of apoptosis‐stimulating protein two of p53 ameliorates acute kidney injury induced by ischemia reperfusion in mice through upregulation of autophagy

Acute kidney injury (AKI) has become a common disorder with a high risk of morbidity and mortality, which remains major medical problem without reliable and effective therapeutic intervention. Apoptosis‐stimulating protein two of p53 (ASPP2) is a proapoptotic member that belongs to p53 binding protein family, which plays a key role in regulating apoptosis and cell growth. However, the role of ASPP2 in AKI has not been reported. To explore the role of ASPP2 in the progression of AKI, we prepared an AKI mouse model induced by ischaemia reperfusion (I/R) in wild‐type (ASPP2^+/+) mice and ASPP2 haploinsufficient (ASPP2^+/?) mice. The expression profile of ASPP2 were examined in wild‐type mice. The renal injury, inflammation response, cellular apoptosis and autophagic pathway was assessed in ASPP2^+/+ and ASPP2^+/? mice. The renal injury, inflammation response and cellular apoptosis was analysed in ASPP2^+/+ and ASPP2^+/? mice treated with 3‐methyladenine or vehicle. The expression profile of ASPP2 showed an increase at the early stage while a decrease at the late stage during renal injury. Compared with ASPP2^+/+ mice, ASPP2 deficiency protected mice against renal injury induced by I/R, which mainly exhibited in slighter histologic changes, lower levels of blood urea nitrogen and serum creatinine, and less apoptosis as well as inflammatory response. Furthermore, ASPP2 deficiency enhanced autophagic activity reflecting in the light chain 3‐II conversion and p62 degradation, while the inhibition of autophagy reversed the protective effect of ASPP2 deficiency on AKI. These data suggest that downregulation of ASPP2 can ameliorate AKI induced by I/R through activating autophagy, which may provide a novel therapeutic strage for AKI.     

Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion

Macrophage migration inhibitory factor (MIF) is pleiotropic cytokine that has multiple effects in many inflammatory and immune diseases. This study reveals a potential role of MIF in acute kidney injury (AKI) in patients and in kidney ischemic reperfusion injury (IRI) mouse model in MIF wild‐type (WT) and MIF knockout (KO) mice. Clinically, plasma and urinary MIF levels were largely elevated at the onset of AKI, declined to normal levels when AKI was resolved and correlated tightly with serum creatinine independent of disease causes. Experimentally, MIF levels in plasma and urine were rapidly elevated after IRI‐AKI and associated with the elevation of serum creatinine and the severity of tubular necrosis, which were suppressed in MIF KO mice. It was possible that MIF may mediate AKI via CD74/TLR4‐NF‐κB signalling as mice lacking MIF were protected from AKI by largely suppressing CD74/TLR‐4‐NF‐κB associated renal inflammation, including the expression of MCP‐1, TNF‐α, IL‐1β, IL‐6, iNOS, CXCL15(IL‐8 in human) and infiltration of macrophages, neutrophil, and T cells. In conclusion, our study suggests that MIF may be pathogenic in AKI and levels of plasma and urinary MIF may correlate with the progression and regression of AKI.     

Autophagy promotes fibrosis and apoptosis in the peritoneum during long‐term peritoneal dialysis

Long‐term peritoneal dialysis is accompanied by functional and histopathological alterations in the peritoneal membrane. In the long process of peritoneal dialysis, high‐glucose peritoneal dialysis solution (HGPDS) will aggravate the peritoneal fibrosis, leading to decreased effectiveness of peritoneal dialysis and ultrafiltration failure. In this study, we found that the coincidence of elevated TGF‐β1 expression, autophagy, apoptosis and fibrosis in peritoneal membrane from patients with peritoneal dialysis. The peritoneal membranes from patients were performed with immunocytochemistry and transmission electron microscopy. Human peritoneal mesothelial cells were treated with 1.5%, 2.5% and 4.25% HGPDS for 24 hrs; Human peritoneal mesothelial cells pre‐treated with TGF‐β1 (10 ng/ml) or transfected with siRNA Beclin1 were treated with 4.25% HGPDS or vehicle for 24 hrs. We further detected the production of TGF‐β1, activation of TGF‐β1/Smad2/3 signalling, induction of autophagy, EMT, fibrosis and apoptosis. We also explored whether autophagy inhibition by siRNA targeting Beclin 1 reduces EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. HGPDS increased TGF‐β1 production, activated TGF‐β1/Smad2/3 signalling and induced autophagy, fibrosis and apoptosis hallmarks in human peritoneal mesothelial cells; HGPDS‐induced Beclin 1‐dependent autophagy in human peritoneal mesothelial cells; Autophagy inhibition by siRNA Beclin 1 reduced EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. Taken all together, these studies are expected to open a new avenue in the understanding of peritoneal fibrosis, which may guide us to explore the compounds targeting autophagy and achieve the therapeutic improvement of PD.     

Pharmacologically active microcarriers influence VEGF‐A effects on mesenchymal stem cell survival

Resistance of transplanted mesenchymal stem cells (MSCs) in post‐ischemic heart is limited by their poor vitality. Vascular‐endothelial‐growth‐factor‐A (VEGF‐A) as such or slowly released by fibronectin‐coated pharmacologically‐active‐microcarriers (FN‐PAM‐VEGF) could differently affect survival kinases and anti‐apoptotic mediator (e.g. Bcl‐2). Therefore VEGF‐A or FN‐PAM‐VEGF could differently enhance cell proliferation, and/or resistance to hypoxia/reoxygenation (H/R) of MSCs. To test these hypotheses MSCs were incubated for 6‐days with VEGF‐A alone or with FN‐PAM‐VEGF. In addition, MSCs pre‐treated for 24‐hrs with VEGF‐A or FN‐PAM‐VEGF were subsequently exposed to H/R (72‐hrs 3% O₂ and 3‐hrs of reoxygenation). Cell‐proliferation and post‐hypoxic vitality were determined. Kinases were studied at 30‐min., 1‐ and 3‐days of treatment. Cell‐proliferation increased about twofold (P < 0.01) 6‐days after VEGF‐A treatment, but by a lesser extent (55% increase) with FN‐PAM‐VEGF (P < 0.05). While MSC pre‐treatment with VEGF‐A confirmed cell‐proliferation, pre‐treatment with FN‐PAM‐VEGF protected MSCs against H/R. In the early phase of treatments, VEGF‐A increased phospho‐Akt, phospho‐ERK‐1/2 and phospho‐PKCε compared to the untreated cells or FN‐PAM‐VEGF. Afterword, kinase phosphorylations were higher with VGEF, except for ERK‐1/2, which was similarly increased by both treatments at 3 days. Only FN‐PAM‐VEGF significantly increased Bcl‐2 levels. After H/R, lactate dehydrogenase release and cleaved Caspase‐3 levels were mainly reduced by FN‐PAM‐VEGF. While VEGF‐A enhances MSC proliferation in normoxia, FN‐PAM‐VEGF mainly hampers post‐hypoxic MSC death. These different effects underscore the necessity of approaches suited to the various conditions. The use of FN‐PAM‐VEGF could be considered as a novel approach for enhancing MSC survival and regeneration in hostile environment of post‐ischemic tissues.     

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re‐establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full‐length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9‐week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell‐based gene therapy provides a new approach to cardiac regeneration.