Dongqi Tang

Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors

Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone single-cell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1- or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia.

Up-regulation of p27kip1 contributes to Nrf2-mediated protection against angiotensin II-induced cardiac hypertrophy

AIMS Nuclear factor erythroid-2-related factor 2 (Nrf2) appears to be a negative regulator of maladaptive cardiac remodelling and dysfunction; however, a potential of the Nrf2-mediated cardiac protection in diverse pathological settings remains to be determined. This study was aimed to explore the role of Nrf2 in angiotensin II (Ang II)-induced cardiac hypertrophy. METHODS AND RESULTS Littermate wild-type (WT) and Nrf2 knockout (Nrf2(-/-)) mice were administered Ang II via osmotic mini-pumps for 2 weeks to induce cardiac hypertrophy. Elevation of blood pressure by the continuous Ang II infusion was comparable between WT and Nrf2(-/-) mice. Relative to WT mice, however, Nrf2(-/-) mice exhibited exaggerated myocardial oxidative stress with an impaired induction of a group of antioxidant genes and increased cardiac hypertrophy in response to the sustained Ang II stimulation. In cultured cardiomyocytes, adenoviral overexpression of Nrf2 shRNA enhanced Ang II-induced reactive oxygen species (ROS) production and protein synthesis, whereas adenoviral overexpression of Nrf2 exerted opposite effects. Moreover, Nrf2 deficiency exacerbated Ang II-induced down-regulation of p27(kip1) expression in the heart via a mechanism of post-transcriptional regulation. In contrast, adenoviral overexpression of Nrf2 increased p27(kip1) protein but not mRNA expression and reversed Ang II-induced down-regulation of p27(kip1) protein expression in cultured cardiomyocytes by suppressing ROS formation. Finally, the enhancement of Ang II-induced hypertrophic growth due to the Nrf2 deficiency was negated by overexpressing p27(kip1) in cultured cardiomyocytes. CONCLUSION The Nrf2-p27(kip1) pathway serves as a novel negative feedback mechanism in Ang II-induced pathogenesis of cardiac hypertrophy, independent of changes in blood pressure.

Irisin promotes human umbilical vein endothelial cell proliferation through the ERK signaling pathway and partly suppresses high glucose-induced apoptosis.

Irisin is a newly discovered myokine that links exercise with metabolic homeostasis. It is involved in modest weight loss and improves glucose intolerance. However, the direct effects and mechanisms of irisin on vascular endothelial cells (ECs) are not fully understood. In the current study, we demonstrated that irisin promoted Human Umbilical Vein Endothelial Cell (HUVEC) proliferation. It was further demonstrated that this pro-proliferation effect was mediated by irisin-induced activation of extracellular signal–related kinase (ERK) signaling pathways. Inhibition of ERK signaling with U0126 decreased the pro-proliferation effect of irisin on HUVECs. It was also demonstrated that irisin reduced high glucose-induced apoptosis by up-regulating Bcl-2 expression and down-regulating Bax, Caspase-9 and Caspase-3 expression. In summary, these results suggested that irisin plays a novel role in sustaining endothelial homeostasis by promoting HUVEC proliferation via the ERK signaling pathway and protects the cell from high glucose-induced apoptosis by regulating Bcl-2,Bax and Caspase expression.

Nrf2 Deficiency Exaggerates Doxorubicin-Induced Cardiotoxicity and Cardiac Dysfunction

The anticancer therapy of doxorubicin (Dox) has been limited by its acute and chronic cardiotoxicity. In addition to a causative role of oxidative stress, autophagy appears to play an important role in the regulation of Dox-induced cardiotoxicity. However, the underlying mechanisms remain unclear. Accordingly, we explored a role of nuclear factor erythroid-2 related factor 2 (Nrf2) in Dox-induced cardiomyopathy with a focus on myocardial oxidative stress and autophagic activity. In wild type (WT) mice, a single intraperitoneal injection of 25 mg/kg Dox rapidly induced cardiomyocyte necrosis and cardiac dysfunction, which were associated with oxidative stress, impaired autophagy, and accumulated polyubiquitinated protein aggregates. However, these Dox-induced adverse effects were exaggerated in Nrf2 knockout (Nrf2−/−) mice. In cultured cardiomyocytes, overexpression of Nrf2 increased the steady levels of LC3-II, ameliorated Dox-induced impairment of autophagic flux and accumulation of ubiquitinated protein aggregates, and suppressed Dox-induced cytotoxicity, whereas knockdown of Nrf2 exerted opposite effects. Moreover, the exaggerated adverse effects in Dox-intoxicated Nrf2 depleted cardiomyocytes were dramatically attenuated by forced activation of autophagy via overexpression of autophagy related gene 5 (Atg5). Thus, these results suggest that Nrf2 is likely an endogenous suppressor of Dox-induced cardiotoxicity by controlling both oxidative stress and autophagy in the heart.

Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation

Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage β-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet β-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.

Irisin Induces Angiogenesis in Human Umbilical Vein Endothelial Cells In Vitro and in Zebrafish Embryos In Vivo via Activation of the ERK Signaling Pathway.

As a link between exercise and metabolism, irisin is assumed to be involved in increased total body energy expenditure, reduced body weight, and increased insulin sensitivity. Although our recent evidence supported the contribution of irisin to vascular endothelial cell (ECs) proliferation and apoptosis, further research of irisin involvement in the angiogenesis of ECs was not conclusive. In the current study, it was found that irisin promoted Human Umbilical Vein Endothelial Cell (HUVEC) angiogenesis via increasing migration and tube formation, and attenuated chemically-induced intersegmental vessel (ISV) angiogenic impairment in transgenic TG (fli1: GFP) zebrafish. It was further demonstrated that expression of matrix metalloproteinase (MMP) 2 and 9 were also up-regulated in endothelial cells. We also found that irisin activated extracellular signal–related kinase (ERK) signaling pathways. Inhibition of ERK signaling by using U0126 decreased the pro-migration and pro-angiogenic effect of irisin on HUVEC. Also, U0126 inhibited the elevated expression of MMP-2 and MMP-9 when they were treated with irisin. In summary, these findings provided direct evidence that irisin may play a pivotal role in maintaining endothelium homeostasis by promoting endothelial cell angiogenesis via the ERK signaling pathway.

Protective Effect of Irisin on Atherosclerosis via Suppressing Oxidized Low Density Lipoprotein Induced Vascular Inflammation and Endothelial Dysfunction.

Irisin, a newly discovered myokine, is considered as a promising candidate for the treatment of metabolic disturbances and cardiovascular diseases. In the present study, we used two animal models, apolipoprotein E-deficient mice fed on a high-cholesterol diet and a mouse carotid partial ligation model to test the anti-atherosclerotic effect of irisin. Irisin treatment (0.5 μg/g body weight/day) significantly reduced the severity of aortic atherosclerosis in apolipoprotein E-deficient mice fed on a high-cholesterol diet and suppressed carotid neointima formation in a carotid partial ligation model. It was associated with decreased inflammation and cell apoptosis in aortic tissues. In addition, in a cell culture model, irisin restored ox-LDL-induced human umbilical vein endothelial cell dysfunction by reducing the levels of inflammatory genes via inhibiting the reactive oxygen species (ROS)/ p38 MAPK/ NF-κB signaling pathway activation and inhibiting cell apoptosis via up-regulating Bcl-2 and down-regulating Bax and caspase-3 expression. Our study demonstrated that irisin significantly reduced atherosclerosis in apolipoprotein E-deficient mice via suppressing ox-LDL-induced cell inflammation and apoptosis, which might have a direct therapeutic effect on atherosclerotic diseases.

Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin’s potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value of exercise, which promotes irisin release.

Deubiquitinating enzyme CYLD mediates pressure overload-induced cardiac maladaptive remodeling and dysfunction via downregulating Nrf2

Ubiquitin proteasome system (UPS) consists of ubiquitin, ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), proteasomes, and deubiquitinating enzymes (DUBs). Ubiquitin, E1s, several E2s, E3s, and proteasomes play an important role in the regulation of cardiac homeostasis and dysfunction; however, less is known about the role of DUBs in the heart. Here, we uncovered a crucial role of cyclindromatosis (CYLD), a DUB, in mediating cardiac maladaptive remodeling and dysfunction. CYLD expression was dramatically upregulated in the cardiomyocytes of hypertrophic and failing human and murine hearts. Knockout of CYLD improved survival rate and alleviated cardiac hypertrophy, fibrosis, apoptosis, oxidative stress, and dysfunction in mice that were subjected to sustained pressure overload induced by transverse aortic constriction. Deep sequencing and gene array analyses revealed that the most dramatically changed genes are those involving in the free radical scavenging pathway and cardiovascular disease, including fos, jun, myc, and nuclear factor erythroid-2 related factor 2 (Nrf2) in the heart. Moreover, knockdown of CYLD enhanced mitogen-activated protein kinase (MAPK) ERK- and p38-mediated expression of c-jun, c-fos, and c-myc, which govern Nrf2 expression in cardiomyocytes. The CYLD deficiency-induced suppression of reactive oxygen species (ROS) formation, death and hypertrophy in cardiomyocytes was blocked by additional knockdown of Nrf2. Taken together, our findings demonstrate for the first time that CYLD mediates cardiac maladaptive remodeling and dysfunction, most likely via enhancing myocardial oxidative stress in response to pressure overload. At the molecular level, CYLD interrupts the ERK- and p38-/AP-1 and c-Myc pathways to suppress Nrf2-operated antioxidative capacity, thereby enhancing oxidative stress in the heart.

Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNFα-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNFalpha)-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNFalpha-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNFalpha hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.