Effects of Systemic Peroxisome Proliferator-Activated Receptor Gamma Inhibition on Bone and Immune Cells in Aged Female Mice.

This study investigates the effects of peroxisome proliferator-activated receptor gamma (PPARγ) inhibition on bone and immune cell profiles in aged female mice, as well as in vitro stromal stem cell osteogenic differentiation and inflammation gene expression. The hypothesis was that inhibition of PPARγ would increase bone mass and alter immune and other cellular functions. Our results showed that treatment with PPARγ antagonist GW9662 for 6 weeks reduced bone volume and trabecular number and increased trabecular spacing. However, inhibition of PPARγ had no significant effect on marrow and spleen immune cell composition in aged female mice. In vitro experiments indicated that GW9662 treatment increased the expression of osteogenic genes but did not affect adipogenic genes. Additionally, GW9662 treatment decreased the expression of several inflammation-related genes. Overall, these findings suggest that PPARγ inhibition may have adverse effects on bone in aged female mice.

Increased Oxidative and Nitrative Stress Accelerates Aging of the Retinal Vasculature in the Diabetic Retina.

Hyperglycemia-induced retinal oxidative and nitrative stress can accelerate vascular cell aging, which may lead to vascular dysfunction as seen in diabetes. There is no information on whether this may contribute to the progression of diabetic retinopathy (DR). In this study, we have assessed the occurrence of senescence-associated markers in retinas of streptozotocin-induced diabetic rats at 8 and 12 weeks of hyperglycemia as compared to normoglycemic aging (12 and 14 months) and adult (4.5 months) rat retinas. We have found that in the diabetic retinas there was an up-regulation of senescence-associated markers SA-ß-Gal, p16INK4a and miR34a, which correlated with decreased expression of SIRT1, a target of miR34a. Expression of senescence-associated factors primarily found in retinal microvasculature of diabetic rats exceeded levels measured in adult and aging rat retinas. In aging rats, retinal expression of senescence associated-factors was mainly localized at the level of the retinal pigmented epithelium and only minimally in the retinal microvasculature. The expression of oxidative/nitrative stress markers such as 4-hydroxynonenal and nitrotyrosine was more pronounced in the retinal vasculature of diabetic rats as compared to normoglycemic aging and adult rat retinas. Treatments of STZ-rats with the anti-nitrating drug FeTPPS (10mg/Kg/day) significantly reduced the appearance of senescence markers in the retinal microvasculature. Our results demonstrate that hyperglycemia accelerates retinal microvascular cell aging whereas physiological aging affects primarily cells of the retinal pigmented epithelium. In conclusion, hyperglycemia-induced retinal vessel dysfunction and DR progression involve vascular cell senescence due to increased oxidative/nitrative stress.

Soluble epoxide hydrolase inhibition and peroxisome proliferator activated receptor γ agonist improve vascular function and decrease renal injury in hypertensive obese rats.

Cardiometabolic syndrome occurs with obesity and consists of pathophysiological factors that increase the risk for cardiovascular events. Soluble epoxide hydrolase inhibition (sEHi) is a novel therapeutic approach that exerts renal and cardiovascular protection. Although sEHi as a therapeutic approach is promising, it could be more effective for the treatment of cardiometabolic syndrome when combined with peroxisome proliferator activated receptor γ (PPARγ) agonists. We hypothesized that the PPARγ agonist, rosiglitazone in combination with a sEHi (tAUCB) will provide synergistic actions to decrease blood pressure, improve vascular function, decrease inflammation, and prevent renal damage in spontaneously hypertensive obese rats (SHROB). SHROB were treated with rosiglitazone, tAUCB or the combination of tAUCB and rosiglitazone for four-weeks and compared with spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Blood pressure increased in SHROB (164 ± 7 mmHg) and decreased 10 mmHg when treated with rosiglitazone, tAUCB, or tAUCB and rosiglitazone. Mesenteric artery dilation to the K(ATP) channel opener pinacidil was attenuated in SHROB (E(Max) = 77 ± 7%), compared with WKY (E(Max) = 115 ± 19) and SHR (E(Max) = 93 ± 12%). Vasodilation to pinacidil was improved by rosiglitazone (E(Max) = 92 ± 14%) but not tAUCB. Renal macrophage infiltration increased in SHROB and significantly decreased with rosiglitazone or tAUCB and rosiglitazone treatment. Albuminuria was increased in SHROB (90 ± 20 mg/d) and was significantly decreased by the combination of tAUCB and rosiglitazone (37 ± 9 mg/d). Glomerular injury in SHROB was also significantly decreased by tAUCB and rosiglitazone. These results indicate that even though sEHi or PPARγ agonist have benefits when used individually, the combination is more beneficial for the multidisease features in cardiometabolic syndrome.

Effect of simvastatin on high glucose- and angiotensin II-induced activation of the JAK/STAT pathway in mesangial cells.

In the current study, we investigated the effect of simvastatin on the ability of high glucose (HG) and ANG II to activate the JAK2-STAT signaling cascade and induce glomerular mesangial cell (GMC) growth. We found that pretreatment with simvastatin significantly inhibited HG- and ANG II-induced collagen IV production, JAK2 activation, and phosphorylation of STAT1 and STAT3 in GMC. We also found that the activation of JAK2 by HG and ANG II was dependent on the Rho family of GTPases. Consistent with these in vitro results, both albumin protein excretion and phosphorylation of JAK2, STAT1, and STAT3 were attenuated in renal glomeruli by administration of simvastatin in a streptozotocin-induced rat model of HG diabetes. This study demonstrates that simvastatin blocks ANG II-induced activation of the JAK/STAT pathway in the diabetic environment, in vitro and in vivo, and, thereby, provides new insights into the molecular mechanisms underlying early diabetic nephropathy.

Aldose reductase pathway mediates JAK-STAT signaling: a novel axis in myocardial ischemic injury.

The aldose reductase pathway has been demonstrated to be a key component of myocardial ischemia reperfusion injury. Previously, we demonstrated that increased lactate/pyruvate ratio, a measure of cytosolic NADH/NAD+, is an important change that drives the metabolic cascade mediating ischemic injury. This study investigated signaling mechanisms by which the aldose reductase pathway mediates myocardial ischemic injury. Specifically, the influence of the aldose reductase pathway flux on JAK-STAT signaling was examined in perfused hearts. Induction of global ischemia in rats resulted in JAK2 activation followed by STAT5 activation. Pharmacological inhibition of aldose reductase or sorbitol dehydrogenase blocked JAK2 and STAT5 activation and was associated with lower lactate/pyruvate ratio and lower protein kinase C activity. Niacin, known to lower cytosolic NADH/NAD+ ratio independent of the aldose reductase pathway inhibition, also blocked JAK2 and STAT5 activation. Inhibition of protein kinase C also blocked JAK2 and STAT5 activation. Transgenic mice overexpressing human aldose reductase exhibited increased JAK2 and STAT5 activation. Pharmacological inhibition of JAK2 reduced ischemic injury and improved functional recovery similar to that observed in aldose reductase pathway inhibited mice hearts. These data, for the first time, demonstrate JAK-STAT signaling by the aldose reductase pathway in ischemic hearts and is, in part, due to changes in cytosolic redox state.

The neuroprotective effect of 2-(3-pyridyl)-1-azabicyclo[3.2.2]nonane (TC-1698), a novel alpha7 ligand, is prevented through angiotensin II activation of a tyrosine phosphatase.

We have recently provided evidence for nicotine-induced complex formation between the alpha7 nicotinic acetylcholine receptor (nAChR) and the tyrosine-phosphorylated enzyme Janus kinase 2 (JAK2) that results in subsequent activation of phosphatidylinositol-3-kinase (PI-3-K) and Akt. Nicotine interaction with the alpha7 nAChR inhibits Abeta (1-42) interaction with the same receptor, and the Abeta (1-42)-induced apoptosis is prevented through nicotine-induced activation of JAK2. These effects can be shown by measuring markers of cytotoxicity, including the cleavage of the nuclear protein poly(ADP-ribose) polymerase (PARP), the induction of caspase 3, or cell viability. In this study, we found that 2-(3-pyridyl)-1-azabicyclo[3.2.2]nonane (TC-1698), a novel alpha7-selective agonist, exerts neuroprotective effects via activation of the JAK2/PI-3K cascade, which can be neutralized through activation of the angiotensin II (Ang II) AT(2) receptor. Vanadate not only augmented the TC-1698-induced tyrosine phosphorylation of JAK2 but also blocked the Ang II neutralization of TC-1698-induced neuroprotection against Abeta (1-42)-induced cleavage of PARP. Furthermore, when SHP-1 was neutralized via antisense transfection, the Ang II inhibition of TC-1698-induced neuroprotection against Abeta (1-42) was prevented. These results support the main hypothesis that states that JAK2 plays a central role in the nicotinic alpha7 receptor-induced activation of the JAK2-PI-3K cascade in PC12 cells, which ultimately contribute to nAChR-mediated neuroprotection. Ang II inhibits this pathway through the AT(2) receptor activation of the protein tyrosine phosphatase SHP-1. This study supports central and opposite roles for JAK2 and SHP-1 in the control of apoptosis and alpha7-mediated neuroprotection in PC12 cells.

Angiotensin II blockade prevents hyperglycemia-induced activation of JAK and STAT proteins in diabetic rat kidney glomeruli.

Clinical and animal studies show that treatment with angiotensin-converting enzyme (ACE) inhibitors or ANG II-receptor antagonists slows progression of nephropathy in diabetes, indicating ANG II plays an important role in its development. We previously reported that hyperglycemia augments both ANG II-induced growth and activation of Janus kinase (JAK)2 and signal transducers and activators of transcription (STAT) proteins in cultured rat mesangial cells. Furthermore, we demonstrated that the tyrosine kinase enzyme JAK2 plays a key role in both ANG II- and hyperglycemia-induced growth in these cells. We hypothesized that the ACE inhibitor captopril and the ANG II-receptor antagonist candesartan would hinder hyperglycemic-induced activation of JAK and STAT proteins in rat glomeruli, demonstrating that ANG II plays an important role in the activation of these proteins in vivo. Adult male Sprague-Dawley rats were given either streptozotocin (STZ; 60 mg/kg iv) or vehicle, and glomeruli were isolated 2 wk later. Activation of JAK and STAT proteins was evaluated by Western blot analysis for specific tyrosine phosphorylation. Groups of rats were given captopril (75-85 mg x kg(-1) x day(-1)), candesartan (10 mg x kg(-1) x day(-1)), or the JAK2 inhibitor AG-490 (5 mg x kg(-1) x day(-1)) for the study's duration. STZ stimulated glomerular phosphorylation of JAK2, STAT1, STAT3, and STAT5. Phosphorylation was reduced in rats treated with captopril, candesartan, and AG-490. Furthermore, both candesartan and AG-490 inhibited STZ-induced increases in urinary protein excretion. In conclusion, our studies demonstrate that hyperglycemia induces activation of JAK2 and the STATs in vivo via an ANG II-dependent mechanism and that these proteins may be involved in the early kidney damage associated with diabetes.

Angiotensin II blocks nicotine-mediated neuroprotection against beta-amyloid (1-42) via activation of the tyrosine phosphatase SHP-1.

We showed recently that nicotine activates the growth-promoting enzyme Janus kinase 2 (JAK2) in PC12 cells and that preincubation of these cells with the JAK2-specific inhibitor AG-490 blocked the nicotine-induced neuroprotection against beta-amyloid (1-42) [Abeta (1-42)]. These results provided direct evidence for linkage between JAK2 and the alpha7 nicotinic acetylcholine receptor-induced neuroprotection in PC12 cells. We also showed that preincubation with angiotensin II (Ang II), functioning via the angiotensin II type 2 (AT2) receptor, blocked both the nicotine-induced activation of JAK2 and its neuroprotection against Abeta (1-42). Recently growth-inhibitory effects of the AT2 receptor have been reported to be mediated by the activation of protein tyrosine phosphatases (PTPases) and that AT2 receptor stimulation is associated with a rapid activation of the PTPase SHP-1 (the cytoplasmic tyrosine phosphatase that contains Src homology 2 domains), a negative regulator of JAK2 signaling. Therefore, the potential biological significance of AT2 receptor-induced effects on both the nicotine-induced activation of JAK2 and its neuroprotection against Abeta (1-42) led us to investigate whether SHP-1 activation could be involved in this process. We found that Ang II induced the activation of SHP-1 and that an antisense against SHP-1 not only augmented the nicotine-induced tyrosine phosphorylation of JAK2 but also blocked the Ang II neutralization of the nicotine-induced neuroprotection. These results demonstrate that nicotine-induced tyrosine phosphorylation of JAK2 and neuroprotection against Abeta (1-42) in PC12 cells are blocked by Ang II via AT2 receptor-induced activation of SHP-1.

S100B-RAGE-mediated augmentation of angiotensin II-induced activation of JAK2 in vascular smooth muscle cells is dependent on PLD2.

Angiotensin II (Ang II), a vasoactive peptide that is also considered a growth factor, has been implicated in both normal and diabetic cellular proliferation. We recently found that activation of janus kinase 2 (JAK2) is essential for the Ang II-induced proliferation of vascular smooth muscle cells (VSMCs) and that high glucose augments Ang II-induced proliferation of VSMCs by increasing signal transduction through activation of JAK2. Here, we demonstrate that S100B, a ligand for the receptor of advanced glycation end products (RAGEs), augmented both Ang II-induced tyrosine phosphorylation of JAK2 and cell proliferation in VSMCs in a receptor-dependent manner. We also found that S100B-RAGE interaction triggered intracellular generation of reactive oxygen species (ROS), VSMC proliferation, and JAK2 tyrosine phosphorylation via activation of phospholipase D (PLD)2. These results provide direct evidence for linkages between PLD2, ROS production, and S100B-RAGE-induced enhancement of Ang II-induced cell proliferation and activation of JAK2 in VSMCs.

High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway.

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.

Inhibition of the Jak/STAT signaling pathway prevents the high glucose-induced increase in tgf-beta and fibronectin synthesis in mesangial cells.

High glucose (HG) causes glomerular mesangial cell (GMC) growth, production of transforming growth factor (TGF)-beta, and increased synthesis of matrix proteins such as fibronectin, contributing to diabetic nephropathy. We recently found that exposure of cells to HG also activates the growth-promoting enzyme janus kinase 2 (JAK2) and its latent signal transducers and activators of transcription (STAT) transcription factors (STAT1, STAT3, and STAT5). Our purpose was to determine the effect that inhibition of JAK2 and these STAT transcription factors has on the HG-induced increase in TGF-beta and fibronectin synthesis in GMC. Exposure of GMC to 25 mmol/l glucose caused the activation of JAK2, STAT1, STAT3, and STAT5 plus an increase in TGF-beta and fibronectin synthesis, as compared with 5.5 mmol/l glucose. This HG-induced increase in synthesis of TGF-beta and fibronectin was prevented by concomitant incubation with AG-490, a specific JAK2 inhibitor. The HG-induced JAK2, STAT1, and STAT3 tyrosine phosphorylations in GMC were also abolished by AG-490. Preincubation of GMC cultured in 25 mmol/l glucose with a specific JAK2 or STAT1 antisense oligonucleotide also prevented both TGF-beta and fibronectin synthesis. These results provide direct evidence for linkages between JAK2, STAT1, and the glucose-induced overproduction of TGF-beta and fibronectin in GMC.

Janus kinase 2, an early target of alpha 7 nicotinic acetylcholine receptor-mediated neuroprotection against Abeta-(1-42) amyloid.

The molecular mechanisms of alpha7 nicotinic acetylcholine receptor (nAChR)-mediated neuroprotection remain unclear. In this study we provide evidence that nicotine stimulation of alpha7 nAChR transduces signals to phosphatidylinositol 3-kinase and Akt via Janus kinase 2 (JAK2) in a cascade, which results in neuroprotection. Exposure to beta-amyloid results in the activation of the apoptotic enzyme caspase-3 and cleavage of the DNA-repairing enzyme poly-(ADP-ribose) polymerase. This cascade is inhibited by nicotine through JAK2 activation, and these effects are blocked by preincubation with the JAK2-specific inhibitor AG-490. We also found that pretreatment of cells with angiotensin II blocks the nicotine-induced activation of JAK2 via the AT(2) receptor and completely prevents alpha7 nAChR-mediated neuroprotective effects further suggesting a pivotal role for JAK2. These findings identify novel mechanisms of receptor interactions relevant to neuronal viability and suggest novel therapeutic strategies to optimize neuroprotection.

Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose.

BACKGROUND: Both high glucose (HG) and angiotensin II (Ang II) causes glomerular mesangial cell (GMC) growth and increased synthesis of matrix proteins like collagen IV contributing to diabetic nephropathy. We have recently found that exposure of vascular smooth muscle cells to HG augments the Ang II activation of the growth promoting JAK/STAT pathway. We hypothesized that Ang II activation of the JAK/STAT pathway is altered by HG in GMC, and that this pathway might be linked to the Ang II-induced growth and overproduction of collagen IV in GMC in HG conditions. METHODS: GMC were cultured under normal glucose (NG; 5.5 mmol/L) and HG (25 mmol/L) for 48 hours and stimulated with Ang II (0.1 micromol/L) for various times. GMC lysate was then immunoprecipitated and/or immunoblotted with SHP-1, SHP-2 and phosphospecific JAK2 and STAT antibodies. The HG and Ang II induced growth and collagen IV synthesis studies were performed in GMC transfected with JAK2 antisense or JAK2 sense. GMC growth was monitored via [3H]-thymidine incorporation, and collagen IV synthesis via ELISA. RESULTS: We found that Ang II-induced JAK2, STAT1, STAT3, STAT5A/B and SHP-2 phosphorylations were enhanced by HG, whereas that of SHP-1 was reduced. Ang II-induced growth and collagen IV synthesis also were increased under HG conditions. Transfection of GMC with JAK2 antisense oligonucleotides blocked the Ang II-induced growth and collagen IV synthesis in both NG and HG conditions. CONCLUSION: These results provide evidence that activation of the JAK/STAT pathway by HG or/and Ang II may be of importance in the increased GMC cell growth and collagen IV synthesis that is seen in diabetic nephropathy.