Lianghu Huang

Palmitate Causes Endoplasmic Reticulum Stress and Apoptosis in Human Mesenchymal Stem Cells: Prevention by AMPK Activator

Elevated circulating saturated fatty acids concentration is commonly associated with poorly controlled diabetes. The highly prevalent free fatty acid palmitate could induce apoptosis in various cell types, but little is known about its effects on human mesenchymal stem cells (MSCs). Here, we report that prolonged exposure to palmitate induces human bone marrow-derived MSC (hBM-MSC) and human umbilical cord-derived MSC apoptosis. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cell apoptosis. Palmitate activated XBP1 splicing, elF2α (eukaryotic translation initiation factor 2α) phosphorylation, and CHOP, ATF4, BiP, and GRP94 transcription in hBM-MSCs. ERK1/2 and p38 MAPK phosphorylation were also induced by palmitate in hBM-MSCs. A selective p38 inhibitor inhibited palmitate activation of the ER stress, whereas the ERK1/2 inhibitors had no effect. The AMP-activated protein kinase activator aminoimidazole carboxamide ribonucleotide blocked palmitate-induced ER stress and apoptosis. These findings suggest that palmitate induces ER stress and ERK1/2 and p38 activation in hBM-MSCs, and AMP-activated protein kinase activator prevents the deleterious effects of palmitate by inhibiting ER stress and apoptosis.

Low-protein diet supplemented with ketoacids reduces the severity of renal disease in 5/6 nephrectomized rats: a role for KLF15

Dietary protein restriction is an important treatment for chronic kidney disease. Herein, we tested the effect of low-protein or low-protein plus ketoacids (KA) diet in a remnant kidney model. Rats with a remnant kidney were randomized to receive normal protein diet (22%), low-protein (6%) diet (LPD), or low-protein (5%) plus KA (1%) diet for 6 months. Protein restriction prevented proteinuria, decreased blood urea nitrogen levels, and renal lesions; however, the LPD retarded growth and decreased serum albumin levels. Supplementation with KA corrected these abnormalities and provided superior renal protection compared with protein restriction alone. The levels of Kruppel-like factor-15 (KLF15), a transcription factor shown to reduce cardiac fibrosis, were decreased in remnant kidneys. Protein restriction, which increased KLF15 levels in the normal kidney, partially recovered the levels of KLF15 in remnant kidney. The expression of KLF15 in mesangial cells was repressed by oxidative stress, transforming growth factor-β, and tumor necrosis factor (TNF)-α. The suppressive effect of TNF-α on KLF15 expression was mediated by TNF receptor-1 and nuclear factor-κB. Overexpression of KLF15 in mesangial and HEK293 cells significantly decreased fibronectin and type IV collagen mRNA levels. Furthermore, KLF15 knockout mice developed glomerulosclerosis following uninephrectomy. Thus, KLF15 may be an antifibrotic factor in the kidney, and its decreased expression may contribute to the progression of kidney disease.

Umbilical Cord Mesenchymal Stromal Cell With Autologous Bone Marrow Cell Transplantation in Established Type 1 Diabetes: A Pilot Randomized Controlled Open-Label Clinical Study to Assess Safety and Impact on Insulin Secretion

OBJECTIVE To determine the safety and effects on insulin secretion of umbilical cord (UC) mesenchymal stromal cells (MSCs) plus autologous bone marrow mononuclear cell (aBM-MNC) stem cell transplantation (SCT) without immunotherapy in established type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS Between January 2009 and December 2010, 42 patients with T1D were randomized (n = 21/group) to either SCT (1.1 × 106/kg UC-MSC, 106.8 × 106/kg aBM-MNC through supraselective pancreatic artery cannulation) or standard care (control). Patients were followed for 1 year at 3-month intervals. The primary end point was C-peptide area under the curve (AUCC-Pep) during an oral glucose tolerance test at 1 year. Additional end points were safety and tolerability of the procedure, metabolic control, and quality of life. RESULTS The treatment was well tolerated. At 1 year, metabolic measures improved in treated patients: AUCC-Pep increased 105.7% (6.6 ± 6.1 to 13.6 ± 8.1 pmol/mL/180 min, P = 0.00012) in 20 of 21 responders, whereas it decreased 7.7% in control subjects (8.4 ± 6.8 to 7.7 ± 4.5 pmol/mL/180 min, P = 0.013 vs. SCT); insulin area under the curve increased 49.3% (1,477.8 ± 1,012.8 to 2,205.5 ± 1,194.0 mmol/mL/180 min, P = 0.01), whereas it decreased 5.7% in control subjects (1,517.7 ± 630.2 to 1,431.7 ± 441.6 mmol/mL/180 min, P = 0.027 vs. SCT). HbA1c decreased 12.6% (8.6 ± 0.81% [70.0 ± 7.1 mmol/mol] to 7.5 ± 1.0% [58.0 ± 8.6 mmol/mol], P < 0.01) in the treated group, whereas it increased 1.2% in the control group (8.7 ± 0.9% [72.0 ± 7.5 mmol/mol] to 8.8 ± 0.9% [73 ± 7.5 mmol/mol], P < 0.01 vs. SCT). Fasting glycemia decreased 24.4% (200.0 ± 51.1 to 151.2 ± 22.1 mg/dL, P < 0.002) and 4.3% in control subjects (192.4 ± 35.3 to 184.2 ± 34.3 mg/dL, P < 0.042). Daily insulin requirements decreased 29.2% in only the treated group (0.9 ± 0.2 to 0.6 ± 0.2 IU/day/kg, P = 0.001), with no change found in control subjects (0.9 ± 0.2 to 0.9 ± 0.2 IU/day/kg, P < 0.01 vs. SCT). CONCLUSIONS Transplantation of UC-MSC and aBM-MNC was safe and associated with moderate improvement of metabolic measures in patients with established T1D.

Simultaneous Islet and Kidney Transplantation in Seven Patients With Type 1 Diabetes and End-Stage Renal Disease Using a Glucocorticoid-Free Immunosuppressive Regimen With Alemtuzumab Induction

OBJECTIVE—The aim of this study was to evaluate the efficiency and safety of simultaneous islet and kidney transplantation in patients with type 1 diabetes and end-stage renal disease using a glucocorticoid-free immunosuppressive regimen with alemtuzumab induction. RESEARCH DESIGN AND METHODS—Seven patients with type 1 diabetes and end-stage renal failure were transplanted with allogenic islets and kidneys procured from brain-dead donors. To prevent organ rejection, patients received alemtuzumab for induction immunosuppression, followed by sirolimus and tacrolimus. No glucocorticoids were given at any time. RESULTS—The median duration of follow-up was 18.3 months (range 13–31). Kidney survival was 100%. Four patients became insulin independent at 1 year. The other three reduced insulin use to less than 25% of the amount required before transplantation. Serum C-peptide levels were significantly greater posttransplant in all patients, indicating continued islet function. No major procedure-related complications were observed. CONCLUSIONS—Our results demonstrate that a steroid-free immunosuppressive regimen consisting of alemtuzumab, sirolimus, and tacrolimus is feasible for simultaneous islet and kidney transplantation. The question of whether this induction regimen is superior to more standard induction deserves large studies.

Increased susceptibility to acute kidney injury due to endoplasmic reticulum stress in mice lacking tumor necrosis factor-α and its receptor 1

Endoplasmic reticulum (ER) stress is actively involved in acute organ injury. Since tumor necrosis factor α (TNFα) plays a role in acute kidney injury, and induces ER stress and cell death in vitro, we examined the contribution of TNFα to acute kidney ER stress induced by tunicamycin. Contrary to expectation, tunicamycin caused much more severe kidney injury in TNFα-/- than in wild-type mice. The major site of kidney injury in TNFα-/- mice was proximal tubules, which showed extensive cell vacuolation, lipid accumulation, and apoptosis. Reconstitution of TNFα-/- mice with TNFα 24 h before tunicamycin injection reversed the susceptibility. When TNFα-receptor-deficient mice were treated with tunicamycin, severe renal injury developed in TNFR1-/- but not TNFR2-/- mice, suggesting this aspect of TNFα action was through TNF receptor-1 (TNFR1). In response to tunicamycin-induced acute ER stress, kidneys from neither TNFα-/- nor TNFR1-/- mice showed a significant increase in phosphorylated eukaryotic translation initiation factor 2α (eIF2α), a key step in ER stress regulation. Moreover, proximal tubular cells from TNFR1-/- mice did not show increased eIF2α phosphorylation in response to tunicamycin and were susceptible to ER stress-induced cell death. Finally, treatment of proximal tubule cells isolated from TNFR1-/- mice with an inhibitor of eIF2α phosphatase increased the levels of phosphorylated eIF2α and substantially reduced tunicamycin-induced cell death. Thus, disruption of TNFR1 signaling leads to dysregulation of eIF2α and increased susceptibility to acute ER stress injury in the kidney.

MicroRNA-mediated epigenetic targeting of Survivin significantly enhances the antitumor activity of paclitaxel against non-small cell lung cancer

Elevated expression of Survivin correlates with poor prognosis, tumor recurrence, and drug resistance in various human cancers, including non-small cell lung cancer (NSCLC). The underlying mechanism of Survivin upregulation in cancer cells remains elusive. To date, no Survivin-targeted therapy has been approved for cancer treatment. Here, we explored the molecular basis resulting in Survivin overexpression in NSCLC and investigated the antitumor activity of the class I HDAC inhibitor entinostat in combination with paclitaxel. Our data showed that entinostat significantly enhanced paclitaxel-mediated anti-proliferative/anti-survival effects on NSCLC cells in vitro and in vivo. Mechanistically, entinostat selectively decreased expression of Survivin via induction of miR-203 (in vitro and in vivo) and miR-542-3p (in vitro). Moreover, analysis of NSCLC patient samples revealed that the expression levels of miR-203 were downregulated due to promoter hypermethylation in 45% of NSCLC tumors. In contrast, increased expression of both DNA methytransferase I (DNMT1) and Survivin was observed and significantly correlated with the reduced miR-203 in NSCLC. Collectively, these data shed new lights on the molecular mechanism of Survivin upregulation in NSCLC. Our findings also support that the combinatorial treatments of entinostat and paclitaxel will likely exhibit survival benefit in the NSCLC patients with overexpression of DNMT1 and/or Survivin. The DNMT1-miR-203-Survivin signaling axis may provide a new avenue for the development of novel epigenetic approaches to enhance the chemotherapeutic efficacy against NSCLC.

miRNA-302 facilitates reprogramming of human adult hepatocytes into pancreatic islets-like cells in combination with a chemical defined media.

The direct conversion of one cell type to another without an intermediate pluripotent stage is required for regenerative therapies. The ventral pancreas and liver share a common developmental origin. Recent studies have shown that hepatocytes could be induced to transdifferentiate into insulin-producing cells. In this paper, we showed a new strategy to achieve the direct conversion of human hepatocytes into surrogate β cells. Hepatocytes were transfected with microRNA-302 (miR-302) mimic and Pdx1, Ngn3 and MafA expressed plasmids, followed by a chemical-defined culture system for maturation of insulin-secreting cells. Co-transfection of miR-302 mimic increased the transcription of pancreatic development-related genes (Sox17, Foxa2, and endogenous Pdx1). Furthermore, at the end of this treatment, hepatocytes became insulin expressed cells that released the hormone in response to a physiological glucose change in vitro. This work shows that miR-302 participation may facilitates the conversion of adult hepatocytes into pancreatic islets-like cells.

CHOP favors endoplasmic reticulum stress-induced apoptosis in hepatocellular carcinoma cells via inhibition of autophagy

C/EBP-homologous protein (CHOP) is an important component of the endoplasmic reticulum (ER) stress response. We demonstrated the induction of ER stress in response to tunicamycin stimulation, as evidenced by increased expression of chaperone proteins Grp78, Grp94, and enhanced eukaryotic initiation factor 2 subunit 1 (eIF2α) phosphorylation in hepatocellular carcinoma cells. Tunicamycin-induced ER stress resulted in apoptosis and autophagy simultaneously. While inhibition of autophagy mediated by 3-methyladenine pretreatment or direct knockdown of LC3B promoted cell apoptosis, activation of autophagy with rapamycin decreased tunicamycin- induced apoptosis in HCC cells. Furthermore, CHOP was shown to be significantly upregulated upon treatment with tunicamycin in HCC cells. Specific knockdown of CHOP not only enhanced tunicamycin-induced autophagy, but also significantly attenuated ER stress-induced apoptosis in HCC cells. Accordingly, simultaneous inhibition of autophagy in HCC cells with CHOP-knockdown could partially resensitize ER stress-induced apoptosis. Taken together, our data indicate that CHOP may favor ER stress-induced apoptosis in HCC cells via inhibition of autophagy in vitro.

Cotransplantation of Bone Marrow Mononuclear Cells and Umbilical Cord Mesenchymal Stem Cells in Avascular Necrosis of the Femoral Head

OBJECTIVE We sought to investigate the therapeutic effects of cotransplantation of autologous bone marrow mononuclear cells (BMMNCs) and allogeneic umbilical cord mesenchymal stem cells (UC-MSCs) on avascular necrosis of the femoral head (ANFH). METHODS In all, 30 patients (49 hips; 24 males and 6 females) with ANFH were enrolled. According to the system of the Association Research Circulation Osseous, there were 24 hips in phase II and 25 hips in phase Ⅲ. Blood supply to the femoral head was evaluated by using digital subtraction angiography. Generally, 60 to 80 mL of autologous BMMNCs and 30 to 50 mL of UC-MSCs were infused into the femoral head artery. Harris scores including pain and joint function were used to evaluate the effects before and 3, 6, 9, and 12 months after transplantation. Computed tomography and radiographs were performed before and 12 months after the treatment. RESULTS Clinical symptoms of pain and claudication were gradually improved. After the treatment, 93.3% (28/30), 86.7% (26/30), and 86.7% (26/30) of patients showed relief of hip pain, improvement of joint function, and extended walking distances, respectively. The Harris scores were increased significantly at 3, 6, and 12 months posttransplant compared with those pretransplant. In addition, the bone lesions in 89.7% of hips (44/49) were improved as showed on computed tomography after transplantation. CONCLUSION Cotransplantation of autologous BMMNCs and allogeneic UC-MSCs showed therapeutic effect on ANFH without severe adverse effects.

Protective effects of mesenchymal stromal cells on adriamycin-induced minimal change nephrotic syndrome in rats and possible mechanisms

BACKGROUND AIMS Minimal change nephrotic syndrome is the most frequent cause of nephrotic syndrome in childhood. Current treatment regimes, which include glucocorticoid hormones and immunosuppressive therapy, are effective and have fast response. However, because of the side effects, long treatment course, poor patient compliance and relapse, novel approaches for the disease are highly desired. METHODS The adriamycin-induced nephrotic rat model was established. Rats were allocated to a model group, a prednisone group or mesenchymal stromal cell (MSC) group. Clinical parameters in each treatment group were determined at 2 weeks, 4 weeks and 8 weeks. The messenger RNA (mRNA) levels of synaptopodin, p21 and monocyte chemoattractant protein-1 were determined through the use of quantitative real-time-polymerase chain reaction. Protein levels were determined by means of Western blot or enzyme-linked immunosorbent assay. Podocytes were isolated and apoptotic rate after adriamycin with or without MSC treatment was analyzed by means of flow cytometry. RESULTS MSC intervention improved renal function as assessed by urinary protein, blood creatinine and triglyceride levels. MSC intervention reduced adriamycin-induced renal tissue damage visualized by immunohistochemistry and light and electron microscopic analysis and reduced adriamycin-induced podocyte apoptosis. After MSC intervention, mRNA and protein levels of synaptopodin and p21 in renal cortex were significantly increased. MSCs also restored synaptopodin mRNA and protein expression in isolated podocytes. In addition, monocyte chemoattractant protein-1 mRNA in renal cortex and protein level in serum of the MSC treatment group were significantly decreased compared with that in the adriamycin-induced nephropathy model group. CONCLUSIONS Our data indicate that MSCs could protect rats from adriamycin-induced minimal change nephrotic syndrome, and the protective effects of MSCs are mediated through multiple actions.