Increase In Pro-inflammatory Cytokines Research Articles

Orexin A increases sympathetic nerve activity through promoting expression of proinflammatory cytokines in Sprague Dawley rats.

Accumulating evidence suggests that orexin signalling is involved in the regulation of blood pressure and cardiovascular function. However, the underlying mechanisms are not clear. Here, we test the hypothesis that upregulated orexin A signalling in the paraventricular nucleus (PVN) increases sympathetic nerve activity (SNA) through stimulating expression of proinflammatory cytokines (PICs). In vivo sympathetic nerve recordings were performed to test the impact of PVN orexin signalling on sympathetic outflow in Sprague Dawley (SD) rats. Real-time PCR was carried out to assess effects of central administration of orexin A on PVN PICs expression in SD rats. To test whether orexin A-induced increases in PICs were exclusively mediated by orexin receptor 1 (OX1R), OX1R-expressing PC12 (PC12-OX1R) cells were incubated with different dose of orexin A, and then, PICs mRNA and immunoreactivity were measured. Orexin A microinjection (25pmol) into the PVN significantly increased splanchnic SNA (93.5%) and renal SNA (83.3%) in SD rats, and these increases were attenuated by OX1R antagonist SB408124. Intracerebroventricular injection of orexin A (0.2nmol) into SD rats increased mRNA levels of PICs including IL-1-β (2.7-fold), IL-6 (1.7-fold) and TNF-α (1.5-fold), as well as Fra1 (1.6-fold) in the PVN. Orexin A treatment in PC12-OX1R cells resulted in a dose- and time-dependent increase in the expression of PICs and Fra1, a subunit of AP1 transcriptional factor. The increase in the PICs was blocked by AP1 blocker curcumin. Paraventricular nucleus orexin system activation is involved in SNA regulation maybe through triggering AP1-PICs pathway.

Inhibition of NF-κB activity in the hypothalamic paraventricular nucleus attenuates hypertension and cardiac hypertrophy by modulating cytokines and attenuating oxidative stress

We hypothesized that chronic inhibition of NF-κB activity in the hypothalamic paraventricular nucleus (PVN) delays the progression of hypertension and attenuates cardiac hypertrophy by up-regulating anti-inflammatory cytokines, reducing pro-inflammatory cytokines (PICs), attenuating nuclear factor-κB (NF-κB) p65 and NAD(P)H oxidase in the PVN of young spontaneously hypertensive rats (SHR). Young normotensive Wistar–Kyoto (WKY) and SHR rats received bilateral PVN infusions with NF–κB inhibitor pyrrolidine dithiocarbamate (PDTC) or vehicle for 4weeks. SHR rats had higher mean arterial pressure and cardiac hypertrophy as indicated by increased whole heart weight/body weight ratio, whole heart weight/tibia length ratio, left ventricular weight/tibia length ratio, cardiomyocyte diameters of the left cardiac ventricle, and mRNA expressions of cardiac atrial natriuretic peptide (ANP) and beta-myosin heavy chain (β-MHC). These SHR rats had higher PVN levels of proinflammatory cytokines (PICs), reactive oxygen species (ROS), the chemokine monocyte chemoattractant protein-1 (MCP-1), NAD(P)H oxidase activity, mRNA expression of NOX-2 and NOX-4, and lower PVN IL-10, and higher plasma levels of PICs and NE, and lower plasma IL-10. PVN infusion of NF-κB inhibitor PDTC attenuated all these changes. These findings suggest that NF-κB activation in the PVN increases sympathoexcitation and hypertensive response, which are associated with the increases of PICs and oxidative stress in the PVN; PVN inhibition of NF-κB activity attenuates PICs and oxidative stress in the PVN, thereby attenuates hypertension and cardiac hypertrophy.

Angiotensin II causes imbalance between pro‐ and anti‐inflammatory cytokines by modulating GSK‐3β in neuronal culture

Emerging evidence indicates that the balance between pro-inflammatory cytokines (PICs) and anti-inflammatory cytokines (AICs) within the brain is an important determinant in the outcome of hypertension. However, the mechanism by which this dysregulation occurs is not known. We aimed to investigate whether AngII induces imbalance between PIC and AIC by modulating downstream transcription factors, NFκB and cyclic AMP response element-binding protein (CREB), and whether AngII-induced effects are mediated by glycogen synthase kinase-3β (GSK-3β). CATH.a neurons were exposed to AngII (10 nM-1 μM) over a preset time course. In another set of experiments, GSK-3β was knock down by using lentivirus containing short hairpin RNA targeting GSK-3β (L-sh-GSK3β) before AngII exposure. Cell extracts were subjected to RT-PCR, immunoblot and immunoprecipitation. AngII caused time-dependent increase in PICs (TNF-α and IL-1β) and reduction in AIC (IL-10). AngII exposure caused reduced phosphorylated CREB(Ser-133) and increased p-NFκB(Ser-276) levels, leading to reduced CREB-CBP and increased NFκB-CBP binding. These results were accompanied by increased activation of GSK-3β, as indicated by increased p-GSK3(Tyr-216) to p-GSK3(Ser-9) ratio. In a subsequent study, pretreatment with L-sh-GSK3β attenuated AngII-induced alterations in PICs and IL-10 by augmenting CREB-CBP and attenuating NFκB-CBP binding. Collectively, these findings are the first to provide direct evidence that AngII-induced dysregulation in cytokines is mediated by GSK-3β-mediated alterations in downstream transcription factors in neuronal cells. Our data also reveal that AngII-induced effects could be alleviated by GSK-3β inhibition, suggesting GSK-3β as an important therapeutic target for hypertension that is characterized by increased PICs and NFκB activation.

Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension

Dysfunction of brain renin-angiotensin system (RAS) components is implicated in the development of hypertension. We previously showed that angiotensin (Ang) II-induced hypertension is mediated by increased production of proinflammatory cytokines (PIC), including tumor necrosis factor (TNF), in brain cardiovascular regulatory centers such as the paraventricular nucleus (PVN). Presently, we tested the hypothesis that central TNF blockade prevents dysregulation of brain RAS components and attenuates Ang II-induced hypertension. Male Sprague-Dawley rats were implanted with radio-telemetry transmitters to measure mean arterial pressure (MAP) and subjected to intracerebroventricular (ICV) infusion of etanercept (10 µg/kg/day) with/without concurrent subcutaneous 4-week Ang II (200 ng/kg/min) infusion. Chronic Ang II infusion resulted in a significant increase in MAP and cardiac hypertrophy, which was attenuated by inhibition of brain TNF with etanercept. Etanercept treatment also attenuated Ang II-induced increases in PIC and decreases in IL-10 expression in the PVN. Additionally, Ang II infusion increased expression of pro-hypertensive RAS components (ACE and AT1R), while decreasing anti-hypertensive RAS components (ACE2, Mas, and AT2 receptors), within the PVN. ICV etanercept treatment reversed these changes. Ang II-infusion was associated with increased oxidative stress as indicated by increased NAD(P)H oxidase activity and super oxide production in the PVN, which was prevented by inhibition of TNF. Moreover, brain targeted TNF blockade significantly reduced Ang II-induced NOX-2 and NOX-4 mRNA and protein expression in the PVN. These findings suggest that chronic TNF blockade in the brain protects rats against Ang II-dependent hypertension and cardiac hypertrophy by restoring the balance between pro- and anti-hypertensive RAS axes and inhibiting PIC and oxidative stress genes and proteins in the PVN.

Central blockade of TLR4 improves cardiac function and attenuates pro‐inflammatory cytokines and oxidative stress in hypertensive rats

Role of pro‐inflammatory cytokines (PICs) and oxidative stress within the paraventricular nucleus (PVN) in the pathogenesis of hypertension (HTN) is well established. Although role of toll‐like receptor 4 (TLR4) in various cardiovascular diseases is becoming evident, role of brain TLR4 in the pathogenesis of HTN has never been explored. We hypothesized that TLR4 upregulation within the PVN contributes to the development of HTN. Male SD rats were infused with Angiotensin II (AngII) (200 ng/kg/min) or vehicle by subcutaneous miniosmotic pumps; a subgroup of rats were given intracerebroventricular infusion of Viral Inhibitory Peptide (VIPER, a specific TLR4 blocker) or control peptide for 14 days. Mean arterial pressure (MAP) and cardiac function were evaluated by radio‐telemetry and echocardiography, respectively. PVN and heart tissues were analyzed by RT‐PCR and western blot. TLR4 expression was dramatically upregulated within the PVN of hypertensive rats. VIPER infusion attenuated MAP and improved cardiac function in hypertensive rats. Interestingly, VIPER infusion prevented the increase in PICs (TNF‐α and IL‐1β) and reduced oxidative stress markers (gp91phox and iNOS) in hypertensive rats. These results suggest that the brain TLR4 plays an important role in hypertensive response by modulating PICs and oxidative stress and that the central blockade of TLR4 exerts beneficial effects in HTN. Funding: LSU

Chronic exercise modulates RAS components and improves balance between pro- and anti-inflammatory cytokines in the brain of SHR

Recently, exercise has been recommended as a part of lifestyle modification for all hypertensive patients; however, the precise mechanisms of its effects on hypertension are largely unknown. Therefore, this study aimed to investigate the mechanisms within the brain that can influence exercise-induced effects in an animal model of human essential hypertension. Young normotensive WKY rats and SHR were given moderate-intensity exercise for 16weeks. Blood pressure was measured bi-weekly by tail-cuff method. Animals were then euthanized; paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM), important cardiovascular regulatory centers in the brain, were collected and analyzed by real-time RT-PCR, Western blot, EIA, and fluorescent microscopy. Exercise of 16-week duration attenuated systolic, diastolic, and mean arterial pressure in SHR. Sedentary SHR exhibited increased pro-inflammatory cytokines (PICs) and decreased anti-inflammatory IL-10 levels in the PVN and RVLM. Furthermore, SHR(sed) rats exhibited elevated levels of ACE, AT1R, and decreased levels of ACE2 and receptor Mas in the PVN and RVLM. Chronic exercise not only prevented the increase in PICs (TNF-α, IL-1β), ACE, and AT1R protein expression in the brain of SHR, but also dramatically upregulated IL-10, ACE2, and Mas receptor expression in SHR. In addition, these changes were associated with reduced plasma AngII levels, reduced neuronal activity, reduced NADPH-oxidase subunit gp91(phox) and inducible NO synthase in trained SHRs indicating reduced oxidative stress. These results suggest that chronic exercise not only attenuates PICs and the vasoconstrictor axis of the RAS but also improves the anti-inflammatory defense mechanisms and vasoprotective axis of the RAS in the brain, which, at least in part, explains the blood pressure-lowering effects of exercise in hypertension.

Angiotensin II induced pro‐inflammatory cytokines and oxidative stress in the brain are attenuated in mice lacking the gene for TNF‐α

Angiotensin II (Ang II) has been shown to have both central and peripheral effects. Recent studies suggest a functional crosstalk between angiotensin II and the pro‐inflammatory cytokine (PIC) tumor necrosis factor‐alpha (TNF‐a) in the progression of cardiovascular diseases. Recent findings from our lab suggest that Ang II increases the expression of PICs in the PVN, and that mice lacking the gene for TNF‐a (TNF‐a−/−) have attenuated hypertensive and hypertrophic responses to Ang II infusion. However, the mechanism for this effect is not known. In this study we examined whether Ang II infusion in TNF‐a−/− mice had any effect on PICs and oxidative stress in the hypothalamus and brainstem. Male TNF‐a−/− and control B61295F2/J (WT) mice were subcutaneously implanted with osmotic minipumps containing Ang II (1µg/kg/min) or saline for 14 days. Ang II infusion resulted in increased expression of PICs such as IL‐1beta, IL‐6, and TNF‐α in the hypothalamus and brainstem of WT mice. Ang II infusion also increased the expression of the NOX‐2 subunit of NADPH oxidase, indicating increased oxidative stress. However, this increase in PICs and oxidative stress was attenuated in Ang II infused TNF‐α ‐/− mice. Findings from the present study suggest that brain inflammatory cytokines contributes to the development of Ang II induced hypertension. Grant support: NHLBI grant HL‐80544 for Dr. Joseph Francis.