Atorvastatin ameliorates diabetic nephropathy through inhibiting oxidative stress and ferroptosis signaling.

Clinically, statins have long been used for the prevention and treatment of chronic renal diseases, however, the underlying mechanisms are not fully elucidated. The present study investigated the effects of atorvastatin on diabetes renal injury and ferroptosis signaling. A mouse model of diabetes was established by the intraperitoneal injection of streptozotocin (50 mg/kg/day) plus a high fat diet with or without atorvastatin treatment. Diabetes mice manifested increased plasma glucose and lipid profile, proteinuria, renal injury and fibrosis, atorvastatin significantly lowered plasma lipid profile, proteinuria, renal injury in diabetes mice. Atorvastatin reduced renal reactive oxygen species (ROS), iron accumulation and renal expression of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), transferrin receptor 1 (TFR1), and increased renal expression of glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor (NRF2) and ferritin heavy chain (FTH) in diabetes mice. Consistent with the findings in vivo, atorvastatin prevented high glucose-induced ROS formation and Fe2+ accumulation, an increase in the expression of 4-HNE, MDA and TFR1, and a decrease in cell viability and the expression of NRF2, GPX4 and FTH in HK2 cells. Atorvastatin also reversed ferroptosis inducer erastin-induced ROS production, intracellular Fe2+ accumulation and the changes in the expression of above-mentioned ferroptosis signaling molecules in HK2 cells. In addition, atorvastatin alleviated high glucose- or erastin-induced mitochondria injury. Ferroptosis inhibitor ferrostatin-1 and antioxidant N-acetylcysteine (NAC) equally reversed the expression of high glucose-induced ferroptosis signaling molecules. Our data support the notion that statins can inhibit diabetes-induced renal oxidative stress and ferroptosis, which may contribute to statins protection of diabetic nephropathy.

Preparation and biological evaluation of antibody targeted metal-organic framework drug delivery system (TDDS) in Her2 receptor-positive cells.

In this study, we designed and prepared a trastuzumab-coupled drug delivery system with pH response characteristics using mesoporous zeolitic imidazolate framework-8 (ZIF-8) as the carrier, Trastuzumab@ZIF-8@DOX. As results, the targeted drug delivery system (TDDS) ultimately showed high drug loading and good biocompatibility. The cumulative curve of drug release indicated that the early leakage levels were low under neutral pH conditions. However, under acidic pH conditions, there was an effective enhancement in drug release, indicating the presence of an explicit pH-triggered drug release mechanism. The results indicate that the prepared nanoparticles have the potential to serve as drug delivery systems, as they can release the loaded drug in a controlled manner. The results of cellular uptake tests showed that the uptake of the nanoparticles was greatly enhanced by the internalization mediated by the HER2 antibody. This finding indicates that the prepared nanoparticles can selectively target cancer cells that overexpress HER2. When the doxorubicin dose was 5 µg/ml, the survival rate of SK-BR-3 cells (cancer cells) was 47.75 %, and the survival rate of HaCaT cells (healthy cells) was 75.25 % when co-cultured with both cells. The therapeutic efficacy of Trastuzumab@ZIF-8@DOX was assessed on BALB/c nude mice to validate its potential as an effective drug delivery system for tumor inhibition in vivo. In conclusion, these findings demonstrate the specificity-targeted and pH-responsive nature of this smart drug delivery system, highlighting its promising prospects for efficient and controllable cancer treatment applications.

The role of angiotensin II activation of yes-associated protein/PDZ-binding motif signaling in hypertensive cardiac and vascular remodeling.

Vascular remodeling is the pathogenic basis of hypertension and end organ injury, and the proliferation of vascular smooth muscle cells (VSMCs) is central to vascular remodeling. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway and crucial for controlling cell proliferation, apoptosis and differentiation. The present study investigated the role of YAP/TAZ in cardiac and vascular remodeling of angiotensin II-induced hypertension. Ang II induced YAP/TAZ activation in the heart and aorta, which was prevented by YAP/TAZ inhibitor verteporfin. Treatment with verteporfin significantly reduced Ang II-induced cardiac and vascular hypertrophy with a mild reduction in systolic blood pressure (SBP), verteporfin attenuated Ang II-induced cardiac and aortic fibrosis with the inhibition of transform growth factor (TGF)ß/Smad2/3 fibrotic signaling and extracellular matrix collagen I deposition. Ang II induced Rho A, extracellular signal-regulated kinase 1/2 (ERK1/2) and YAP/TAZ activation in VSMCs, either Rho kinase inhibitor fasudil or ERK inhibitor PD98059 suppressed Ang II-induced YAP/TAZ activation, cell proliferation and fibrosis of VSMCs. Verteporfin also inhibited Ang II-induced VSMC proliferation and fibrotic TGFß1/Smad2/3 pathway. These results demonstrate that Ang II activates YAP/TAZ via Rho kinase/ERK1/2 pathway in VSMCs, which may contribute to cardiac and vascular remodeling in hypertension. Our results suggest that YAP/TAZ plays a critical role in the pathogenesis of hypertension and end organ damage, and targeting the YAP/TAZ pathway may be a new strategy for the prevention and treatment of hypertension and cardiovascular diseases.

Growth hormone-releasing hormone agonist attenuates vascular calcification in diabetic db/db mice.

Introduction: Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. VC increases mortality of all-causes. VC is one of most common cardiovascular complications in type II diabetes. So far, no therapy has been proven to be effective in treatment of clinical VC. The present study investigated the therapeutic effects of MR409, an agonistic analog of growth hormone-releasing hormone (GHRH-A), on VC in diabetic db/db mice. Method and result: Diabetic mice were injected with MR409 subcutaneously every day for 8 weeks. Long-term treatment with MR409 improved serum lipid profile and endothelium-dependent relaxation to acetylcholine, and reduced vascular structural injury in diabetic mice without affecting serum growth hormone level. Echocardiography showed that calcium plaques present in heart valve of diabetic mice disappeared in diabetic mice after treatment with MR409. MR409 inhibited vascular calcium deposition associated with a marked reduction in the expressions of osteogenic-regulated alkaline phosphatase (ALP) and transcription osteogenic marker gene Runx2 in diabetic mice. MR409 also inhibited vascular reactive oxygen species (ROS) generation and upregulated the expressions of anti-calcifying protein Klotho in diabetic mice. Discussion: Our results demonstrate that GHRH-A MR409 can effectively attenuate VC and heart valve calcification, and protect against endothelial dysfunction and vascular injury in diabetic mice without significantly affecting pituitary-growth hormone axis. The mechanisms may involve upregulation of anti-calcifying protein Klotho and reduction in vascular ROS and the expression of redox sensitive osteogenic genes Runx2 and ALP. GHRH-A may represent a new pharmacological strategy for treatment of VC and diabetics associated cardiovascular complications.

Myeloid Angiotensin II Type 1 Receptor Mediates Macrophage Polarization and Promotes Vascular Injury in DOCA/Salt Hypertensive Mice.

Activation of the renin-angiotensin system has been implicated in hypertension. Angiotensin (Ang) II is a potent proinflammatory mediator. The present study investigated the role of myeloid angiotensin type 1 receptor (AT1R) in control of macrophage phenotype in vitro and vascular injury in deoxycorticosterone acetate (DOCA)/salt hypertension. In human THP-1/macrophages, Ang II increased mRNA expressions of M1 cytokines and decreased M2 cytokine expressions. Overexpression of AT1R further increased Ang II-induced expressions of M1 cytokines and decreased M2 cytokines. Silenced AT1R reversed Ang II-induced changes in M1 and M2 cytokines. Ang II upregulated hypoxia-inducible factor (HIF)1α, toll-like receptor (TLR)4, and the ratio of pIκB/IκB, which were prevented by silenced AT1R. Silenced HIF1α prevented Ang II activation of the TLR4/NFκB pathway. Furthermore, Ang II increased HIF1α via reactive oxygen species-dependent reduction in prolyl hydroxylase domain protein 2 (PHD2) expression. The expressions of AT1R and HIF1α and the ratio of pIκB/IκB were upregulated in the peritoneal macrophages of DOCA hypertensive mice, and the specific deletion of myeloid AT1R attenuated cardiac and vascular injury and vascular oxidative stress, reduced the recruitment of macrophages and M1 cytokine expressions, and improved endothelial function without significant reduction in blood pressure. Our results demonstrate that Ang II/AT1R controls the macrophage phenotype via stimulating the HIF1α/NFκB pathway, and specific myeloid AT1R KO improves endothelial function, vascular inflammation, and injury in salt-sensitive hypertension. The results support the notion that myeloid AT1R plays an important role in the regulation of the macrophage phenotype, and dysfunction of this receptor may promote vascular dysfunction and injury in salt-sensitive hypertension.

Oxytocin-induced endothelial nitric oxide dependent vasorelaxation and ERK1/2-mediated vasoconstriction in the rat aorta.

Oxytocin is a neuropeptide produced primarily in the hypothalamus and plays an important role in the regulation of mammalian birth and lactation. It has been shown that oxytocin has important cardiovascular protective effects. Here we investigated the effects of oxytocin on vascular reactivity and underlying the mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and in rat aorta ex vivo. Oxytocin increased phospho-eNOS (Ser 1177) and phospho-Akt (Ser 473) expression in HUVECs in vitro and the aorta of rat ex vivo. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited oxytocin-induced Akt and eNOS phosphorylation. In the rat aortic rings, oxytocin induced a biphasic vascular reactivity: oxytocin at low dose (10-9-10-8 M) initiated a vasorelaxation followed by a vasoconstriction at high dose (10-7 M). L-NAME (a nitric oxide synthase inhibitor), endothelium removal or wortmannin abolished oxytocin-induced vasorelaxation, and slightly enhanced oxytocin-induced vasoconstriction. Atosiban, an oxytocin/vasopressin 1a receptor inhibitor, totally blocked oxytocin-induced relaxation and vasoconstriction. PD98059 (ERK1/2 inhibitor) partially inhibited oxytocin-induced vasoconstriction. Oxytocin also increased aortic phospho-ERK1/2 expression, which was reduced by either atosiban or PD98059, suggesting that oxytocin-induced vasoconstriction was partially mediated by oxytocin/V1aR activation of ERK1/2. The present study demonstrates that oxytocin can activate different signaling pathways to cause vasorelaxation or vasoconstriction. Oxytocin stimulation of PI3K/eNOS-derived nitric oxide may participate in maintenance of cardiovascular homeostasis, and different vascular reactivities to low or high dose of oxytocin suggest that oxytocin may have different regulatory effects on vascular tone under physiological or pathophysiological conditions.

Agonistic analog of growth hormone-releasing hormone promotes neurofunctional recovery and neural regeneration in ischemic stroke.

Ischemic stroke can induce neurogenesis. However, most stroke-generated newborn neurons cannot survive. It has been shown that MR-409, a potent synthetic agonistic analog of growth hormone-releasing hormone (GHRH), can protect against some life-threatening pathological conditions by promoting cell proliferation and survival. The present study shows that long-term treatment with MR-409 (5 or 10 µg/mouse/d) by subcutaneous (s.c.) injection significantly reduces the mortality, ischemic insult, and hippocampal atrophy, and improves neurological functional recovery in mice operated on for transient middle cerebral artery occlusion (tMCAO). Besides, MR-409 can stimulate endogenous neurogenesis and improve the tMCAO-induced loss of neuroplasticity. MR-409 also enhances the proliferation and inhibits apoptosis of neural stem cells treated with oxygen and glucose deprivation-reperfusion. The neuroprotective effects of MR-409 are closely related to the activation of AKT/CREB and BDNF/TrkB pathways. In conclusion, the present study demonstrates that GHRH agonist MR-409 has remarkable neuroprotective effects through enhancing endogenous neurogenesis in cerebral ischemic mice.

Activation of Yes-Associated Protein/PDZ-Binding Motif Pathway Contributes to Endothelial Dysfunction and Vascular Inflammation in AngiotensinII Hypertension.

Yes-associated protein (YAP) and its associated coactivator of PDZ-binding motif (TAZ) are co-transcriptional regulators and down effectors of the Hippo signaling pathway. Recent studies have shown that the Hippo/YAP signaling pathway may play a role in mediating vascular homeostasis. This study investigated the role of YAP/TAZ in endothelial dysfunction and vascular inflammation in angiotensin (Ang)II hypertensive mice. The infusion of AngII (1.1 mg/kg/day by mini-pump) for 3 weeks induced the activation of YAP/TAZ, manifested by decreased cytosolic phosphor-YAP and phosphor-TAZ, and increased YAP/TAZ nuclear translocation, which were prevented by YAP/TAZ inhibitor verteporfin. AngII significantly increased systolic blood pressure (SBP), macrophage infiltration, and expressions of proinflammatory cytokines, and impaired endothelial function in the aorta of the mice. Treatment with verteporfin improved endothelial function and reduced vascular inflammation with a mild reduction in SBP. AngII also induced YAP/TAZ activation in human umbilical vein endothelial cells in vitro, which were prevented by LB-100, an inhibitor of protein phosphatase 2A (PP2A, a major dephosphorylase). Treatment with LB-100 reversed AngII-induced proinflammatory cytokine expression and impairment of phosphor-eNOS expression in vitro. Our results suggest that AngII induces YAP/TAZ activation via PP2A-dependent dephosphorylation, which may contribute to the impairment of endothelial function and the induction of vascular inflammation in hypertension. YAP/TAZ may be a new target for hypertensive vascular injury.

Macrophage depletion protects against endothelial dysfunction and cardiac remodeling in angiotensin II hypertensive mice.

Objective: Hypertension is associated with a low-grade systemic inflammation in cardiovascular system. Macrophage infiltration may initiate an inflammatory process that contributes to vascular and ventricular remodeling in hypertensive human and mice. The present study investigated the effect of chemical depletion of macrophage using liposome encapsulated clodronate (LEC) on cardiac hypertrophy and remodeling in angiotensin (Ang) II hypertensive mice.Methods: C57BL/6 mice received an Ang II (1.1 mg/kg/day with a minipump) infusion for 2 weeks to induce hypertension. Endothelium-dependent relaxation (ED) was examined by organ bath, hematoxylin and staining and Masson-Trichrome staining were used to evaluate aorta and cardiac hypertrophy and fibrosis.Results: Ang II infusion significantly increased systolic blood pressure (SBP), cardiac hypertrophy and fibrosis, and impaired EDR accompanied by increased macrophage infiltration in the heart. Treatment with LEC significantly lowered Ang II-induced cardiac hypertrophy and fibrosis and cardiac macrophage infiltration, and improved EDR with a mild reduction in SBP. Ang II increased the expression of inflammatory cytokines tumor necross factor alpha and interleukin 1 beta and profibrotic factors transforming growth factor beta 1 and fibronectin in the heart, with was reduced by LEC treatment. Treatment with LEC prevented Ang II-induced the phosphorphorylation of ERK1/2 and c-Jun-N-terminal kinase.Conclusions: Our study suggests that cardiac macrophage may be critical for hypertensive cardiac hypertrophy and remodeling, the underlying mechanisms may involve initial heart inflammation and the activation of hypertrophic MAPKs pathway.

Inhibition of YAP activation attenuates renal injury and fibrosis in angiotensin II hypertensive mice.

The Hippo/YAP (yes-associated protein) pathway is an important signaling pathway to control organ development and tissue homeostasis. YAP is a downstream effector of the Hippo pathway and a critical mediator of mechanic stress. Hypertensive nephropathy is characterized with glomerular sclerosis stiffness and renal fibrosis. The present study investigated the role of YAP pathway in angiotensin (Ang) II hypertensive renal injury by using YAP activation inhibitor verteporfin. Ang II increased the protein expression of YAP in renal nucleus fraction, decreased phospho-YAP, and phospho-LATS1/2 (large tumor suppressors 1 and 2) expressions in renal cytoplasmic fraction, suggesting Ang II activation of renal YAP. Ang II significantly increased systolic blood pressure (SBP), proteinuria, glomerular sclerosis, and fibrosis; treatment with verteporfin attenuated Ang II-induced proteinuria and renal injury with a mild reduction in SBP. Moreover, Ang II increased the protein expressions of inflammatory factors including tumor necrosis factor α, interleukin 1ß, and monocyte chemoattractant protein-1, and profibrotic factors including transforming growth factor ß, phospho-Smad3 and fibronectin. Verteporfin reversed abovementioned Ang II-induced molecule expressions. Our results for the first time demonstrate that the activation of the YAP pathway promotes hypertensive renal inflammation and fibrosis, which may promote hypertensive renal injury. YAP may be a new target for prevention and treatment of hypertensive renal diseases.

CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage.

Background and Purpose: CAPN1 (calpain1)­an intracellular Ca2+-regulated cysteine protease­can be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined. Methods: In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucose­deprived primary neuron in vitro model. Results: CAPN1 was activated in the cortex of permanent middle cerebral occlusion­operated rats and oxygen-glucose deprivation­exposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage. Conclusions: The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemia­mediated autophagy-lysosomal pathway defects and neuronal damage.

Nicotine, smoking, podocytes, and diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. Besides glycemic and blood pressure control, environmental factors such as cigarette smoking (CS) adversely affect the progression of DN. The effects of CS on DN progression have been attributed to combustion-generated molecules without consideration to the role of nicotine (NIC), responsible for the addictive properties of both CS and electronic cigarettes (ECs). Podocytes are essential to preserve the structure and function of the glomerular filtration barrier, and strong evidence indicates that early podocyte loss promotes DN progression. We performed experiments in human podocytes and in a mouse model of diabetes that develops nephropathy resembling human DN. We determined that NIC binding to podocytes in concentrations achieved with CS and ECs activated NADPH oxidase, which sets in motion a dysfunctional molecular network integrated by cyclooxygenase 2, known to induce podocyte injury; downregulation of AMP-activated protein kinase, important for maintaining cellular energy stores and antioxidation; and upregulation of CD36, which increased lipid uptake and promoted apoptosis. In diabetic mice, NIC increased proteinuria, a recognized marker of chronic kidney disease progression, accompanied by reduced glomerular podocyte synaptopodin, a crucial stabilizer of the podocyte cytoskeleton, and increased fibronectin expression. This novel study critically implicates NIC itself as a contributor to DN progression in CS and EC users.NEW & NOTEWORTHY In this study, we demonstrate that nicotine increases the production of reactive oxygen species, increases cyclooxygenase-2 expression, and upregulates Cd36 while inducing downregulation of AMP-activated protein kinase. In vivo nicotine increases proteinuria and fibronectin expression in diabetic mice. This study demonstrates that effects of nicotine on podocytes are responsible, at least in part, for the deleterious effects of smoking in the progression of chronic kidney disease, including diabetic nephropathy.

Macrophage Depletion Improves Endothelial Insulin Resistance and Protects against Cardiovascular Injury in Salt-Sensitive Hypertension.

Vascular endothelial insulin signaling is critical for the maintenance of vascular and metabolic homeostasis. We have previously shown that in hypertensive Dahl rats, impaired vascular insulin action is linked to angiotensin II activation of the NFκB inflammatory pathway. Macrophage polarization (M1) has implicated in hypertensive and metabolic diseases. Here, we investigated the effect of macrophage depletion using liposome-encapsulated clodronate (LEC) on endothelial insulin resistance and cardiovascular remodeling in Dahl salt-sensitive (DS) rats. High salt intake (HS) for 5 weeks increased systolic blood pressure (SBP: 192 ± 5 vs. 144 ± 4 mmHg in NS, p < 0.05), aortic and cardiac hypertrophy, cardiac fibrosis, and impaired acetylcholine- and insulin-induced vasorelaxation, accompanied by impaired insulin activation of endothelial nitric oxide synthases (eNOS)/NO signaling. HS rats had a significant increase in CD68 (a monocyte/macrophage marker) expression in the aorta and the heart. LEC reduced SBP (168 ± 5 mmHg, p < 0.05) and cardiovascular injury and improved acetylcholine- and insulin-mediated vasorelaxation and insulin signaling molecules with a reduction in the macrophage infiltration in the aorta and the heart. HS rats also manifested an increase in the aortic expressions of inflammatory cytokines, including the ratio of phosphorylated inhibitory kappa B (Iκb)/Iκb, tumor necrosis factor α, and phosphorylated c-Jun N-terminal kinase (JNK) and oxidative stress, which were reduced in HS/LEC rats. Our results suggest that in salt-sensitive hypertension, macrophage may importantly contribute to endothelial insulin resistance, vascular inflammation, and injury. These findings support the idea that macrophages may be a new target for immunotherapy of vasculopathy in hypertensive and metabolic disorders.

Tumor Necrosis Factor Alpha Deficiency Improves Endothelial Function and Cardiovascular Injury in Deoxycorticosterone Acetate/Salt-Hypertensive Mice.

It has been shown that the inflammatory cytokine tumor necrosis factor α (TNFα) plays a role in the development of hypertension and end-stage renal diseases. We hypothesize that TNFα contributes to endothelial dysfunction and cardiac and vascular injury in deoxycorticosterone acetate (DOCA)/salt-hypertensive mice. The wild-type or TNFα-deficient mice were uninephrectomized and implanted with DOCA pellet treatment for 5 weeks; the mice were given either tap water or 1% NaCl drinking water. DOCA mice developed hypertension (systolic blood pressure (SBP): 167 ± 5 vs. 110 ± 4 mmHg in control group, p < 0.05), cardiac and vascular hypertrophy, and the impairment of endothelium-dependent relaxation to acetylcholine (EDR). TNFα deficiency improved EDR and lowered cardiac and vascular hypertrophy with a mild reduction in SBP (152 ± 4 vs. 167 ± 5 mmHg in DOCA group, p < 0.05) in DOCA mice. The mRNA expressions of the inflammatory cytokines, including TNFα, interleukin 1ß (IL1ß), monocyte chemotactic protein 1 (MCP1), and monocyte/macrophage marker F4/80 were significantly increased in the aorta of DOCA-hypertensive mice; TNFα deficiency reduced these inflammatory gene expressions. DOCA-hypertensive mice also exhibited an increase in the vascular oxidative fluorescence intensities, the protein expressions of gp91phox and p22phox, and the fibrotic factors transforming growth factor ß and fibronectin. TNFα deficiency reduced oxidative stress and fibrotic protein expressions. The DOCA mice also showed a decrease in the protein expression of eNOS associated with increased miR155 expression; TNFα deficiency prevented a decrease in eNOS expression and an increase in miR155 expression in DOCA mice. These results support the idea that TNFα significantly contributes to vascular inflammation, vascular dysfunction, and injury in hypertension.

Corrigendum: Resistin-Induced Endoplasmic Reticulum Stress Contributes to the Impairment of Insulin Signaling in Endothelium.

[This corrects the article DOI: 10.3389/fphar.2018.01226.].

Resistin-Induced Endoplasmic Reticulum Stress Contributes to the Impairment of Insulin Signaling in Endothelium.

Background: Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity, insulin resistance and cardiovascular diseases (CVDs). Impairment of insulin vascular action may represent a mechanism linking insulin resistance and CVDs. The present study tested the hypothesis that adipocyte-derived resistin inhibits insulin-stimulated endothelial NO production through the induction of ER stress. Methods and Results: Human umbilical vein endothelial cells (HUVC) were incubated with tunicamycin (an inducer of ER stress, 1-20 µg/mL) or resistin (10-100 ng/mL) for 1 h. Either tunicamycin or resistin increased GRP78 (an ER stress marker) expression associated with the impairment of insulin-stimulated Akt/eNOS phosphorylation, which were prevented by TUDCA (an ER stress suppressor). Resistin increased reactive oxygen species (ROS) production, antioxidant treatment inhibited resistin-induced GRP78 expression and impairment of insulin Akt/eNOS signaling, suggesting that ROS may involve resistin-induced ER stress. Resistin also increased JNK phosphorylation, which was prevented by TUDCA. JNK inhibitor SP600125 relieved the resistin inhibitory effects on endothelial insulin Akt/eNOS signaling. In ex vivo experiments, the incubation of aortic rings with resistin impaired insulin- but not acetylcholine-induced vasodilation, which was restored by TUDCA. LNAME (a NOS inhibitor) abolished insulin-induced vasorelaxation in the control or the resistin-treated aortic rings. In addition, resistin increased the mRNA expressions of proinflammatory cytokines tumor nuclear factor (TNF)α and interleukin (IL)-1ß, which were also prevented by TUDCA. Conclusion: Our results support the ideal that ER stress may play an important role for resistin impairment of vascular insulin signaling and insulin action. The mitigation of ER stress may represent a new strategy for prevention and treatment of CVDs in obesity and insulin resistant-related diseases.

Macrophage Depletion Lowered Blood Pressure and Attenuated Hypertensive Renal Injury and Fibrosis.

Monocyte/macrophage recruitment is closely associated with the degree of hypertensive renal injury. We investigated the direct role of macrophages using liposome-encapsulated clodronate (LEC) to deplete monocytes/macrophages in hypertensive renal injury. C57BL/6 mice were treated with a pressor dose of angiotensin (Ang, 1.4 mg/kg/day) II plus LEC or the PBS-liposome for 2 weeks. Ang II mice developed hypertension, albuminuria, glomerulosclerosis, and renal fibrosis. LEC treatment reduced systolic blood pressure (SBP), albuminuria, and protected against renal structural injury in Ang II mice. Ang II significantly increased renal macrophage infiltration (MOMA2+ cells) and the expression of renal tumor necrosis factor α and interleukin ß1, which were significantly reduced in Ang II/LEC mice. Ang II increased renal oxidative stress and the expression of profibrotic factors transforming growth factor (TGF) ß1 and fibronectin. Ang II also inhibited the phosphorylation of endothelial nitric oxide synthase [phospho-endothelial nitric oxide synthesis (eNOS), ser1177]. LEC treatment reduced renal oxidative stress and TGFß1 and fibronectin expressions, and increased phospho-eNOS expression in the Ang II mice. In Dahl rats of salt-sensitive hypertension, LEC treatment for 4 weeks significantly attenuated the elevation of SBP induced by high salt intake and protected against renal injury and fibrosis. Our results demonstrate that renal macrophages play a critical role in the development of hypertension and hypertensive renal injury and fibrosis; the underlying mechanisms may be involved in the reduction in macrophage-driven renal inflammation and restoration of the balance between renal oxidative stress and eNOS. Therefore, macrophages should be considered as a potential therapeutic target to reduce the adverse consequences of hypertensive renal diseases.

Oral nicotine aggravates endothelial dysfunction and vascular inflammation in diet-induced obese rats: Role of macrophage TNFα.

Obesity and cigarette smoke are major cardiovascular (CV) risk factors and, when coexisting in the same individuals, have additive/synergistic effects upon CVD. We studied the mechanisms involved in nicotine enhancement of CVD in Sprague Dawley rats with diet-induced obesity. The rats were fed either a high fat (HFD) or normal rat chow diet with or without nicotine (100 mg/L in drinking water) for 20 weeks. HFD rats developed central obesity, increased systolic blood pressure (SBP), aortic superoxide (O2-) production, and impaired endothelial nitric oxide synthase (eNOS) and endothelium-dependent relaxation to acetylcholine (EDR). Nicotine further increased SBP, O2- and impaired eNOS and EDR in obese rats. In the peritoneal macrophages from obese rats, tumor necrosis factor (TNF) α, interleukin 1ß and CD36 were increased, and were further increased in nicotine-treated obese rats. Using PCR array we found that 3 of 84 target proinflammatory genes were increased by 2-4 fold in the aorta of obese rats, 11 of the target genes were further increased in nicotine-treated obese rats. HUVECs, incubated with conditioned medium from the peritoneal macrophages of nicotine treated-obese rats, exhibited reduced eNOS and increased NADPH oxidase subunits gp91phox and p22phox expression. Those effects were partially prevented by adding anti-TNFα antibody to the conditioned medium. Our results suggest that nicotine aggravates the CV effects of diet-induced obesity including the oxidative stress, vascular inflammation and endothelial dysfunction. The underlying mechanisms may involve in targeting endothelium by enhancement of macrophage-derived TNFα.

Puerarin Improves Vascular Insulin Resistance and Cardiovascular Remodeling in Salt-Sensitive Hypertension.

Puerarin is an isoflavonoid isolated from the Chinese herb, Kudzu roots (also known as Gegen), which has been widely used for the treatment of hypertensive diseases and diabetic mellitus in traditional Chinese medicine. Dahl salt-sensitive (DS) rat is a genetic model of salt-sensitive hypertension with cardiovascular injury and vascular insulin resistance. Here, we investigated whether puerarin improved vascular insulin resistance and attenuated cardiac and aortic remodeling in salt-sensitive hypertension. DS rats were given a normal (NS) or high salt diet (HS) for five weeks. An additional group of DS rats was pretreated with puerarin and NS for 10 days, then switched to HS plus puerarin for five weeks. HS for five weeks increased systolic blood pressure (SBP), cardiac hypertrophy and fibrosis, and aortic hypertrophy with increased the expression of phosphor-ERK1/2 in the aorta and heart; puerarin attenuated cardiac and aortic hypertrophy, cardiac fibrosis and phosphor-ERK1/2 with a mild reduction in SBP. Hypertensive rats also manifested impairment of acetylcholine- and insulin-mediated vasorelaxation and insulin-mediated Akt and eNOS phosphorylation associated with the activation of NF[Formula: see text]B/TNF[Formula: see text]/JNK pathway. Puerarin improved acetylcholine- and insulin-mediated vasorelaxation and insulin-stimulated Akt/NO signaling with the inhibition of the NF[Formula: see text]B inflammatory pathway. Our results demonstrated that in salt-sensitive hypertension, puerarin improved vascular insulin action with cardiovascular beneficial effects. Our results found that the underlying mechanisms may involve its inhibition of NF[Formula: see text]B/JNK and ERK1/2 pathway. These results suggest that puerarin could be used as a new antihypertensive agent to expand our armamentarium for the prevention and treatment of end-organ damage in individuals with hypertension and metabolic diseases.

Puerarin protects against endothelial dysfunction and end-organ damage in Ang II-induced hypertension.

Puerarin, a major isoflavonoid compound from Chinese herb Kudzu roots, has been widely used for the treatment of hypertensive and cardiovascular diseases in China. Here, we investigated puerarin's beneficial effects on the cardiovascular system in angiotensin (Ang) II-induced hypertensive rats. Sprague-Dawley rats were treated with Ang II for 5 days or with puerarin for 10 days followed by Ang II and puerarin for 5 days. Endothelium-dependent relaxation (EDR) to acetylcholine was determined using an organ chamber bath. Ang II increased the systolic blood pressure (SBP: 178 ± 5 mmHg vs. 112 ± 3 mmHg in control, p < 0.05), aortic (30%, p < 0.05), and left ventricular (LV) weight (23%); puerarin reduced SBP (160 ± 2 mmHg, p < 0.05), aortic, and left ventricular weight in Ang II-infused rats. Puerarin also reduced aortic medial thickness and myocardial cell surface area in Ang II-infused rats. Compared with control rats, Ang II infused rats exhibited an impaired EDR with reduction in the protein expression of phosphor-eNOS at Ser 1177 and an increase in the expression of gp91phox (85%), p22phox (113%), transforming growth factor ß1 (145%) and vascular cell adhesion molecule 1 (82%). Puerarin improved EDR and reversed the changes in Ang II-induced protein expression of above molecules. Our results demonstrate that in Ang II-induced hypertensive rats, puerarin protects against endothelial dysfunction and end organ damage with a mild reduction in SBP, and that the cardiovascular beneficial effects of puerarin may be in part attributed to its anti-oxidant and upregulation of phosphor-eNOS.