Involvement of Tumor Necrosis Factor-� in AngiotensinII–Mediated Effects on Salt Appetite, Hypertension, and

Cardiac HypertrophySrinivas Sriramula, Masudul Haque, Dewan S.A. Majid, Joseph Francis

Abstract—Hypertension is considered a low-grade inflammatory condition induced by various proinflammatory cytokines,including tumor necrosis factor (TNF)-�. Recent studies have implicated an involvement of TNF-� in the developmentof salt-sensitive hypertension induced by angiotensin II (Ang II). To understand further the relationship between TNF-�and Ang II, we examined the responses to Ang II in TNF-� knockout (TNF-��/�) mice in the present study. Acontinuous infusion of Ang II (1 �g/kg per minute) for 2 weeks was given to both TNF-��/� and wild-type (WT) micewith implanted osmotic minipumps. Daily measurement of water intake, salt intake, and urine output were performedusing metabolic cages. Blood pressure was monitored continuously with implanted radiotelemetry. Ang II administra-tion for 2 weeks caused increases in salt (0.2�0.07 to 5.6�0.95 mL/d) and water (5.4�0.34 to 11.5�1.2 mL/d) intakeand in mean arterial pressure (115�1 to 151�3 mm Hg) in wild-type mice, but these responses were absent in TNF-��/�

mice (0.2�0.04 to 0.3�0.09 mL/d, 5.5�0.2 to 6.1�0.07 mL/d, and 113�2 to 123�3 mm Hg, respectively). Cardiachypertrophy induced by Ang II was significantly attenuated in TNF-��/� mice compared with wild-type mice. In a groupof TNF-��/� mice, when replacement therapy was made with recombinant TNF-�, Ang II induced similar responses insalt appetite, mean arterial pressure, and cardiac hypertrophy, as observed in wild-type mice. These results suggest thatTNF-� plays a mechanistic role in mediating chronic Ang II–induced effects on salt appetite and blood pressure, as wellas on cardiac hypertrophy. (Hypertension. 2008;51:1345-1351.)

Key Words: angiotensin II � TNF-� � salt appetite � cardiac hypertrophy � hypertension

Angiotensin (Ang) II, the effector peptide of the renin-angiotensin system (RAS), plays a key role in regulation

of body fluid homeostasis, the development of hypertension,and the maintenance of cardiovascular function.1,2 Ang II iswidely recognized for its vasoconstrictor effect, therebyregulating vascular tone and systemic blood pressure,3 andexerts its actions by binding to G protein–coupled receptors,angiotensin type 1 (AT1), and angiotensin type 2 (AT2), locatedon the plasma membrane of target cells throughout the body.4,5

The AT1 plays a predominant role in the central regulation ofarterial blood pressure and cardiovascular remodeling.3,4

Ang II has been shown to have both central and peripheraleffects. In the peripheral vasculature, it normally acts to raisearterial pressure by AT1–mediated vasoconstrictor effects.This pressor response of Ang II administration is also knownto be partially modulated by the concomitant release ofendothelin, prostaglandins, NO, superoxide, and other freeradicals from endothelial cells.6 Ang II also contributes tocardiac and vascular remodeling through its direct effect onthe heart and the blood vessels.2,7,8 In addition, Ang IIstimulates aldosterone, which acts on the renal distal tubules

and collecting ducts to retain sodium and water, therebyraising blood pressure.9 Centrally, Ang II plays an importantrole in regulating the salt appetite and thirst mediated byAT1.10–12 Apart from these, Ang II also acts as a growth factorand stimulator of proinflammatory cytokines, such as tumornecrosis factor (TNF)-�, interleukin-6, and chemokines.13–15

TNF-� is a multifunctional cytokine that plays an impor-tant role in diverse physiological and pathophysiologicalprocesses, such as inflammation, cell survival, growth, dif-ferentiation, and apoptosis.16,17 Because inflammation is akey component in the pathogenesis of hypertension andcardiovascular disease, the interaction between Ang II andTNF-� may play an important role in the modulation ofhypertensive response. Several in vitro and in vivo studiessuggest the existence of cross-talk between the RAS andTNF-�.13,18–20 For instance, Ang II treatment induces theproduction of TNF-� in cultured cardiomyocytes and fibro-blasts.13,21 In addition, TNF-� treatment increased AT1

mRNA levels in neonatal rat cardiac fibroblasts.22 Adminis-tration of the AT1 receptor antagonist valsartan inhibited theexpression of TNF-� in a murine model of arterial injury.23 In

Received October 2, 2007; first decision October 17, 2007; revision accepted March 6, 2008.From the Comparative Biomedical Sciences (S.S., M.H., J.F.), School of Veterinary Medicine, Louisiana State University, Baton Rouge; the

Department of Pharmacology (J.F.), Louisiana State University Health Sciences Center, New Orleans; and the Department of Physiology (D.S.A.M.),Tulane University Health Sciences Center, New Orleans, La.

Correspondence to Joseph Francis, Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, 1909 Skip BertmanDr, Baton Rouge, LA 70803. E-mail jfrancis@lsu.edu

© 2008 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.107.102152

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patients with hypertension or heart failure, chronic blockadeof AT1 resulted in a significant decrease in the circulatinglevels of TNF-�.24,25 More importantly, blockade of TNF-�by etanercept has been shown to prevent renal damage ingenetically hypertensive rats and to lower blood pressure inrats with hypertension induced by Ang II and salt, suggestinga role for TNF-� in blood pressure regulation and renalinjury.26,27 A recent study showed that mice treated withetanercept prevented the hypertension and blunted the in-crease in superoxide production in response to Ang II.28

However, the functional importance of TNF-� in Ang II–induced responses is not yet clearly defined. Therefore, in thepresent study, we examined the role of TNF-� in themediation of Ang II–induced responses, particularly its ef-fects on salt appetite, thirst, and blood pressure, as well as itsrole in myocardial cell growth. The effects of chronicadministration of Ang II have been evaluated in TNF-�knockout mice and compared with those responses in wild-type (WT) control mice to dissect out the role played byTNF-� in the Ang II–induced effects.

MethodsAn expanded Methods section can be found in the online datasupplement, available at http://hyper.ahajournals.org.

Experimental AnimalsTwelve-week-old male B6129S-Tnftm1Gkl/J TNF-� knockout (TNF-��/�) mice and control B6129SF2/J (WT) mice (Jackson Laborato-ries, Bar Harbor, Maine) weighing between 25 and 30 g were usedin this study. The mice were housed in a temperature-controlledroom (23�2°C) with a 12:12 hour light-dark cycle from 7 AM to 7 PM

in the animal quarters. The studies were performed in accordancewith the National Institutes of Health Guidelines for the Care andUse of Laboratory Animals. The experimental procedures wereapproved by the Louisiana State University Institutional Animal Careand Use Committee. They were randomly divided into different groupsaccording to chronic treatment with or without Ang II. Osmoticminipumps were implanted SC to deliver Ang II (1 �g/kg perminute; Sigma Chemical) for 14 days. The control animals wereimplanted with sterile saline pumps. These groups are as follows: (1)WT (sham-operated control); (2) WT�Ang II (WT treated with chronicAng II); (3) TNF-��/� (sham-operated control); (4) TNF-��/��Ang II(knockout mice treated with chronic Ang II); and (5) TNF-��/��Ang II�TNF-� (TNF-�–replaced knockout mice treated with chronic Ang II).In this group of 6 TNF-��/� mice, along with Ang II infusion, humanrecombinant TNF-� was given IP at a dose of 10 ng/g per day for 14days. It should be noted here that we have not seen any sign ofincreasing susceptibility to infection in these TNF-��/� mice inpreoperative or postoperative periods of surgical intervention fortelemetry probes and minipump implantations.

Blood Pressure MeasurementsIn 1 set of experiments, mean arterial pressure in conscious mice wasmeasured using a radiotelemetry system with carotid arterial cathe-ters (Model TA11PA-C10, Data Sciences Intl). Mice were allowed torecover from the surgery for 7 to 10 days before experiments werebegun. Data were collected, stored, and analyzed using DataquestA.R.T. software (Data Sciences Intl).

Metabolic Cage StudyIn another set of experiments, mice were individually housed inspecially designed metabolic cages that prevented food and fecalcontamination of urine samples. Food and water were available adlibitum. Mice were given both water and salt (1.8% sodium chloride)solution in 2 separate receptacles and were allowed to adapt to themetabolic cages for 7 days. After the acclimatization period, daily

water intake, salt intake, and urine volume were measured at baselineand during the 14-day Ang II infusion period. At the end of 14 days,the mice were euthanized and the organs were weighed, and thehypothalamus and left ventricular tissues were collected for mRNAand protein measurements.

EchocardiographyTransthoracic echocardiography was performed on mice underisoflurane anesthesia using a Toshiba Aplio SSH770 (ToshibaMedical) fitted with a 12-MHz transducer at baseline, and after 14days of Ang II infusion. Left ventricular internal dimensions at endsystole and end diastole (LVS and LVD), and interventricular septalwall thickness at the end of systole and at the end of diastole weremeasured digitally on the M-mode tracings and averaged from �3cardiac cycles. Left ventricular fractional shortening was calculatedas follows: [(LVD�LVS)/LVD]�100.

Protein Analysis by Western BlotThe protein expression in the heart and hypothalamus was analyzedby Western blot with the use of anti-AT1 antibody (Santa Cruz).Protein extracts (25 �g) were separated by electrophoresis on 10%SDS-polyacrylamide gels and transferred to polyvinylidene fluoridemembranes (Immobilon-P, Millipore) by electroblotting. Immunore-active bands were visualized through the use of enhanced chemi-luminescence and quantified by VersaDoc MP 5000 System(Bio-Rad).

Analysis of mRNA Expression by Real-Time PCRTotal RNA was isolated from left ventricular tissue and the hypo-thalamus using TRIzol reagent (Invitrogen) according to the manu-facturer’s specifications. cDNA was synthesized from 1 �g of RNAwith an iScript cDNA synthesis kit (Bio-Rad). Real-time PCRamplification reactions were performed with iQ SYBR Green Supermix with ROX (Bio-Rad) in triplicate using the ABI Prism 7900real-time PCR machine (Applied Biosystems). Gene expression wasmeasured by ��CT method and was normalized to 18S ribosomalRNA or GAPDH mRNA levels. The data are presented as the foldexpressions of the gene of interest relative to their control animals.

Statistical AnalysisData were analyzed by ANOVA, followed by Student t test. P�0.05was considered significant.

ResultsBlood Pressure MeasurementsContinuous radiotelemetry recordings of arterial pressureshowed that there were no significant differences in baselineblood pressure measurements among the groups (Figure 1). Ang IIinfusion for 14 days significantly increased mean arterial pres-sure in WT mice from 115�1 to 151�3 mm Hg (P�0.001) butnot in TNF-��/� mice (113�2 to 123�3 mm Hg). However,when TNF-��/� mice were given replacement therapy withhuman recombinant TNF-�, Ang II administration caused anincrease in mean arterial pressure (109�1 to 153�3 mm Hg;P�0.001), similar to that noted in WT mice.

Metabolic ParametersAt baseline, there were no significant differences in salt andwater intakes and urine volumes between the WT andTNF-��/� groups. As illustrated in Figure 2, Ang II infusionfor 14 days in WT mice caused significant increases in saltand water intake, as well as urine output, the increases ofwhich were seen as early as day 5 of infusion. However, thesalt and water intakes and urine output remained unchangedin the TNF-��/� mice infused with Ang II (Figure 2).

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Interestingly, the same treatment in the TNF-��/� micereceiving replacement therapy of human recombinant TNF-�resulted in increases in salt intake, water intake, and urineoutput, noted especially in week 2 of the Ang II infusion.

EchocardiographyEchocardiography was performed to evaluate the effect ofAng II on left ventricular function in these WT and TNF-��/�

mice (Table). The Ang II infusion significantly increasedcardiac wall thickness and end-diastolic and end-systolicdimensions and decreased fractional shortening in WT mice.Conversely, in TNF-��/� mice, wall thickness, end-diastolicand end-systolic dimensions, or fractional shortening werenot affected by Ang II infusion. These results indicated thatcardiac function was well preserved in TNF-��/� mice.

Cardiac Hypertrophy Responses toAng II InfusionTo evaluate changes in the cardiac weight in these AngII–treated WT and TNF-��/� mice, the hearts were harvestedand weighed at the end of the 2-week experimental period.The ratio of heart weight:body weight is calculated. Figure 3illustrated these results on cardiac weight. There were nodifferences in vehicle-treated TNF-��/� and WT mice. In WTmice, Ang II infusion had increased heart weight and alsoincreased the ratio between the heart weight and body weight.In contrast, the TNF-��/� mice with Ang II infusion did notshow any increase in the heart weight:body weight ratio(Figure 3A). To further verify the attenuated hypertrophicresponse in TNF-��/� mice, mRNA levels of atrial natriureticpeptide in the left ventricle were measured by RT-PCR.Although the infusion of Ang II significantly increasedmyocardial levels of atrial natriuretic peptide in WT mice,this upregulation of atrial natriuretic peptide was significantlyattenuated in TNF-��/� mice (Figure 3B).

Gene Expression StudiesTo determine whether the infusion of Ang II alters theexpression of AT1 receptors, we examined the mRNA levels

of AT1 in the heart and hypothalamus by real-time PCR. AT1

mRNA expression was also assessed in the samples collectedfrom WT (n�5 to 6) and TNF-��/� mice (n�5 to 6) beforeAng II infusion. The basal level of AT1 mRNA expressionwas not significantly different between the groups. The AT1

mRNA expression in both heart and hypothalamus wassignificantly increased in Ang II–infused WT mice, whereasthat in Ang II–infused TNF-��/� mice remained unchanged(Figure 4). These results were confirmed at protein levels bythe Western blot analysis (Figure 5). Because both Ang II andTNF-� have been shown to act through the nuclear factor �B(NF-�B)–mediated pathways, we analyzed the P50 subunit ofNF-�B mRNA expression using real-time PCR. Ang IIinfusion significantly increased the NF-�B mRNA expressionin WT mice but not in TNF-��/� mice (Figure 4C). Todetermine which TNF receptor is involved in Ang II infusion,the expression of TNF type 1 and type 2 in the left ventricle

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Figure 1. Effect of Ang II on mean arterial pressure. Mean arteri-al pressure was measured using radiotelemetry for the period ofAng II infusion. Values are means�SEMs (n�6 animals pergroup). P�0.05 vs *WT group and #TNF-��/� group.

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Figure 2. Effect of Ang II on water (A) and salt (1.8% NaCl)intake (B) and on urine excretion (C). Ang II was infused fromday 1 to day 14. Values are means�SEMs (n�6 animals pergroup). P�0.05 vs *WT group and #TNF-��/� group.

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was analyzed. After Ang II infusion, there was a significantincrease in TNF type 1 mRNA and protein expression in theWT but not in the TNF-��/� mice. However, there was nosignificant difference in the TNF type 2 mRNA expression(Figure S1).

DiscussionThe present study demonstrated that the responses to chronicAng II administration on salt and water intake, blood pres-sure, and cardiac function were markedly attenuated in micelacking the gene for the proinflammatory cytokine, TNF-�(TNF-��/� mice). However, these Ang II responses had beenrestored in TNF-��/� mice when these mice were given areplacement therapy with human recombinant TNF-�. Inaddition, it was also observed that the mRNA levels of theAT1 receptor, as well as NF-�B mRNA expressions in theheart and hypothalamus, were increased in response tochronic Ang II in WT but not in TNF-��/� mice. These resultssuggest that a concomitant generation of TNF-� is involvedin the complete expression of Ang II-induced salt appetiteand hypertensive, as well as cardiac hypertrophic responses,possibly via the TNF-�–induced upregulation of AT1 recep-tors, as well as activation of NF-�B activity.

Most of the known physiological actions of Ang II, such asvasoconstriction, increased aldosterone secretion, increasedsympathetic nerve activity, and increased water and sodiumintake, are mediated by the activation of AT1 receptors, whichare widely distributed in all organs, including the liver,

adrenal glands, brain, lung, kidney, heart, and blood ves-sels.4,29 AT1 receptors in the brain are linked to vasopressorresponses, along with regulation of salt appetite, thirst, andmodulation of vasopressin release.12,30,31 Salt appetite andthirst are central nervous system phenomenon. Injection ofAng II into the brain or into the periphery increases saltappetite and thirst in rodents.10,32,33 Ang II is a relatively largepeptide, and it does not readily cross the blood-brain barrier.The central nervous system effect of the Ang II could be via thecircumventricular organ, where the blood-brain barrier is weakor absent.12 These include the organum vasculosum laminaterminalis, subfornical organ, and area postrema. Ablation of thearea postrema or organum vasculosum lamina terminalis regionsattenuates the Ang II–induced response on salt appetite andthirst.12 Thus, all of the components of the RAS system areexpressed within the central nervous system, thereby facilitatingsome of the Ang II–induced effect observed centrally.

Activation of the RAS and the subsequent increase in thelocal production of Ang II is one of the main mechanismsresponsible for hypertension and the progression of cardio-vascular disease. Ang II has been shown in many reports toincrease the expression of various cytokines and chemokinesthat induce cardiac hypertrophy, inflammation, and vascularremodeling that result in the long-term regulation of bloodpressure.2,3 Several studies have shown that blockade of theRAS by ANG-converting enzyme inhibitors or by AT1

receptor blockers attenuates hypertensive response and endorgan damage, as well as inflammatory markers, in many

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Figure 3. Effect of Ang II on cardiac hy-pertrophy. A, Heart weight:body weightratio in WT and TNF-��/� mice. B, mRNAexpression of atrial natriuretic peptide inthe left ventricle. Values aremeans�SEMs. P�0.05 vs *WT groupand #TNF-��/� group.

Table. Echocardiographic Analysis of Cardiac Hypertrophy and Function

Parameters WT WT�Ang II TNF-��/� TNF-��/��Ang II TNF-��/��Ang II�TNF-�

N 6 6 6 6 6

IVSD, mm 0.45�0.03 0.54�0.02* 0.40�0.03 0.44�0.02 0.67�0.06†

IVSS, mm 0.60�0.04 0.81�0.08* 0.65�0.03 0.70�0.03 0.87�0.05†

LVD, mm 3.50�0.10 4.28�0.22* 3.14�0.07 3.17�0.06 3.69�0.07†

LVS, mm 2.55�0.08 3.03�0.14* 2.22�0.05 2.27�0.06 2.73�0.12†

PWD, mm 0.50�0.03 0.57�0.02* 0.42�0.05 0.46�0.02 0.60�0.04†

PWS, mm 0.63�0.07 0.74�0.03* 0.60�0.04 0.67�0.04 0.80�0.10†

FS, % 29.18�1.36 23.57�0.62* 27.77�0.90 27.84�1.06 24.40�0.75†

HR, bpm 422�12 470�33 455�27 423�7.5 472�20

Values are means�SEMs. IVSD and IVSS indicates interventricular septal thickness at end diastole and end systole, respectively; LVD and LVS, left ventricularinternal diameter at end diastole and end systole, respectively; PWD and PWS, posterior wall thickness at end diastole and end systole, respectively; HR, heart rate.

P�0.05 vs *WT group and †TNF-��/� group.

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cardiovascular diseases.34–36 It can be argued that the atten-uated cardiac hypertrophy induced by Ang II in TNF-��/�

mice could be the result of reduced blood pressure responsein those animals and, thus, poses a potential limitation to datainterpretation in the present study. Further studies are neededto examine the pressure-dependent and -independent compo-nents of the attenuated hypertrophic response in these TNF-��/�

mice. However, accumulating evidence from clinical andexperimental studies indicates that there is a functionalcrosstalk between Ang II and several proinflammatory cyto-kines, including TNF-� and interleukin-6, in the regulation ofcardiovascular function.13,18–20 Although there is consider-able evidence from previous studies supporting a role for AT1

activation in Ang II–mediated hypertension, there have beenvery limited studies that examined the functional importanceof TNF-� in Ang II–induced hypertension and AT1 expres-sion. The results from our present study demonstrated a moreclear assessment of the functional involvement of the proin-flammatory cytokine TNF-� in the chronic Ang II–inducedeffects, particularly on salt appetite, blood pressure, andcardiac hypertrophy. However, it may be argued that thecomponent of RAS may be altered, which may influence theresponsiveness of Ang II in these TNF-��/� mice. However,this possibility may be unlikely, because we have observedthat the basal tissue AT1 mRNA expression in both the heartand hypothalamus was not different between these TNF-��/�

mice and WT mice. Further studies are required to define thevarious components of RAS in TNF-��/� mice.

It is well known that Ang II, by its direct effect on theactivation of immune cells, induces the production of inflam-matory mediators, such as TNF-�, and contributes to tissuedamage in hypertensive response.13–15 Recently, blockade ofTNF-� using etanercept has been shown to prevent renaldamage in genetically hypertensive rats and to lower bloodpressure in rats with hypertension induced by Ang II and salt,suggesting a role for TNF-� in blood pressure regulation andrenal injury.26,27 Thus, an interaction between Ang II andTNF-� has been suggested to play an important role inhypertensive response.26,28,37 A recent study by Guzik et al28

showed that Ang II infusion caused infiltration of T lympho-cytes in the aortic adventitia and periaortic fat, increasedT-lymphocyte production of TNF-�, increased vascular su-peroxide production, and led to hypertension in mice. Treat-ment with the TNF-� antagonist etanercept prevented thehypertension and the increase in vascular superoxide causedby Ang II.28 Collectively, these data suggest the notion thatchronic Ang II caused infiltration of T lymphocytes invarious organ systems, including the cardiovascular andcentral nervous system, that facilitates the production ofTNF-�. This enhancement of TNF-� production resulted inupregulation of AT1 receptors to further enhance the directactions of Ang II in the target organs. In addition to thesedirect actions of Ang II, there are also effects of TNF-�–mediated enhanced oxidative stress induced by activation ofreduced nicotinamide-adenine dinucleotide phosphate oxi-dase, possibly via activation of NF-�B activity.13,28

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Figure 4. Effect of Ang II on mRNA expression. A, AT1 receptor in left ventricle. B, AT1 receptor in hypothalamus. C, NF-�B in left ven-tricle. Values are means�SEMs. P�0.05 vs *WT group and #TNF-��/� group.

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Figure 5. Effect of Ang II on proteinexpression of AT1 receptor (AT1R). Repre-sentative Western blot and densitometricanalysis of AT1R protein in left ventricle (A)and hypothalamus (B). *P�0.05 vs WTgroup.

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However, earlier studies had reported differing resultsregarding the role of TNF-� in the regulation of bloodpressure. It had been shown that TNF-� opposed the vaso-constrictor effects of phenylephrine in rat aortic ring prepa-rations.38 Ferreri et al37 demonstrated that the administrationof anti–TNF-� antiserum causes additional increases in meanarterial pressure in a model of Ang II–induced hypertension,indicating that TNF-� may oppose the pressor actions of Ang II.However, a study by Alexander et al39 showed that infusionof TNF-� at a dose of 50 ng/d for 5 days into virgin rats hadno significant effect on blood pressure, but it produced ahypertensive response in pregnant animals. A recent study28

showed that mice treated with etanercept prevented thehypertension and blunted the increase in superoxide produc-tion in response to Ang II. These results also suggest that aninteraction between TNF-� and other factors, includingoxidative stress, is required for full expression of this cyto-kine-induced hypertensive response.

Interaction between the RAS and TNF-� in vivo in cardiachypertrophy was apparent when losartan, an AT1 blocker, wasgiven to transgenic mice overexpressing TNF-� in the heart.Losartan prevented the development of hypertrophy, whereasvehicle treatment produced a significant increase in the heartweight:body weight ratio and LV wall thickness in transgenicmice overexpressing TNF-�.40 Ang II has been shown toinduce TNF-� biosynthesis in the heart by activating NF-�B,which, in turn, induces various proinflammatory cytokinesand chemokines, including TNF-�.13,41 Sustained applicationof TNF-� induces an increase in AT1 mRNA levels in cardiacfibroblasts, which is dependent on NF-�B activation.22,42 AngII, on binding with its receptor, becomes internalized, result-ing in the activation of its intracellular signaling mechanism.AT1 receptor–mediated cellular signaling events have beenpostulated to occur via the G�q mechanism. Interestingly,one of the downstream signaling mechanisms of G�q alsoinvolves NF-�B activation. Similarly, TNF-� production alsoinvolves NF-�B translocation into the nucleus resulting in theperpetuation of TNF-� and other proinflammatory cytokines.In support of the present finding, it has been shown thatNF-�B inhibition attenuates hypertensive response and end-organ damage in spontaneously hypertensive rats.43 Clearly,further studies are needed to understand the molecular mech-anism involved in the TNF-� and RAS interaction.

The present results may provide a beneficial therapeuticimplication of the TNF-� blocker in hypertensive patientswho are also suffering from arthritis. At this moment, nodirect clinical report is available that may indicate thattreatment with a TNF-� blocker may cause a decrease inblood pressure in arthritic patients or have any additivehypotensive effects with the blockers of RAS. Obviously,more comprehensive studies would be required to examinethe therapeutic benefit of a TNF-� blocker in the manage-ment of hypertension and/or arthritis in patients.

In conclusion, the results from the present study suggestthat a concomitant generation of TNF-� is required for thefull expression of chronic Ang II–induced effects, such as saltappetite, hypertension, and cardiac hypertrophy, possibly viaits action in upregulating AT1 receptors, as well as enhancingNF-�B activity.

PerspectivesThe findings of the present study emphasize an importantmechanistic role of TNF-� in the mediation of hypertensiveand cardiac hypertrophy responses induced by chronic Ang IIadministration. These results demonstrate an existence of acrosstalk between the RAS and the proinflammatory cyto-kines in the regulation of cardiovascular and other organfunctions. However, the specific mechanisms and the down-stream signaling pathways by which these 2 systems interactwith each other are not yet clearly defined. These presentfindings provide an important clue in our quest in understand-ing the pathophysiology of hypertension and other cardiovas-cular diseases. Thus, it is imperative that further emphasisshould be focused on complete elucidation of the interactiverole of the RAS and proinflammatory cytokines to increaseour understanding of cardiovascular diseases that are linkedwith inflammatory process.

Sources of FundingThese studies were supported by National Heart, Lung, and BloodInstitute grant HL-80544 and Louisiana Board of Regents LouisianaEducation Quality Support Fund grant to J.F. and by National Heart,Lung, and Blood Institute grant HL-66432 to D.S.A.M.

DisclosuresNone.

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Sriramula et al TNF-� in Ang II–Mediated Effects 1351

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Srinivas Sriramula, Masudul Haque, Dewan S.A. Majid and Joseph FrancisAppetite, Hypertension, and Cardiac Hypertrophy

Mediated Effects on Salt− in Angiotensin IIαInvolvement of Tumor Necrosis Factor-

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2008 American Heart Association, Inc. All rights reserved.

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INVOLVEMENT OF TNF-α IN ANGIOTENSIN II MEDIATED EFFECTS ON SALT

INTAKE, BLOOD PRESSURE AND CARDIAC HYPERTROPHY

Srinivas Sriramula1, Masudul Haque

1, Dewan S.A. Majid

3 and Joseph Francis

1,2

1Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State

University, Baton Rouge, LA-70803; 2Department of Pharmacology, LSU Health Sciences

Center, New Orleans, LA-70119; 3Department of Physiology, Tulane University Health

Sciences Center, New Orleans, LA-70112.

Running Title: TNF-α in ANG II mediated effects

Corresponding Author: Joseph Francis, B.V.Sc., M.V.Sc., Ph.D,

Comparative Biomedical Sciences,

School of Veterinary Medicine,

Louisiana State University,

1909 Skip Bertman Drive,

Baton Rouge, LA 70803, USA

225 578-9752 -Telephone

225 578-9895 -FAX

E-mail: jfrancis@lsu.edu

1

DATA SUPPLEMENT

EXPANDED METHODS

Blood Pressure Measurements Using Telemetry Probes

Blood pressure in conscious mice was measured using a radio telemetry system with carotid

arterial catheters (Data Sciences Intl, DSI; MN). For the implantation of the radiotransmitter,

mice were anaesthetized with a ketamine-xylazine mixture (90 and 10 mg/kg, ip). A midline

skin incision 2cm long from chin to manubrium was performed to isolate the carotid artery.

The catheter portion of the telemetric probe (Model TA11PA-C10, DSI) was inserted into the

ascending aorta through the left carotid artery, and the body of the probe was placed

subcutaneously on the right flank. Mice were placed on a 12-hour light/dark cycle and

received food and water ad libitum throughout the study. Mice were allowed to recover for 7-

10 days before experiments were begun. Data was collected, stored, and analyzed using

Dataquest A.R.T. software (DSI). Only animals giving stable records were included in the

final analysis.

Protein Analysis by Western Blot

Protein was extracted from heart and hypothalamus tissue with lysis buffer. The total protein

concentration in samples was measured by Bio-Rad Dc protein assay. Protein extracts (25µg)

were combined with an equal volume of 2X Laemmli loading buffer, boiled for 5 minutes,

and separated by electrophoresis on 10% SDS-Polyacrylamide gels. Then proteins were

transferred from the gel to PVDF membranes (Immobilon-P, Millipore, Bedford, MA) by

electroblotting. Membranes were blocked with 1% casein in PBS-T for 1 hour and then

incubated with anti-AT1 antibody or anti-TNFR1 antibody (Santa Cruz) overnight at 4°C.

Membranes were washed four times in wash buffer (1X TBS, 0.1% Tween-20), followed by

2

incubation with peroxidase-labeled anti-rabbit IgG for 1 h at room temperature. Membranes

were washed four times with wash buffer at room temperature before the antigen-antibody

complexes were detected by an enhanced chemiluminescence kit (ECL Plus, Amersham).

Autoradiographs were scanned and analyzed by densitometry using VersaDoc MP 5000

System (Bio-Rad). Protein expression of GAPDH was used to check equal loading. All

primary antibodies were used at a dilution of 1:1000 and secondary antibodies were used at a

dilution of 1:10,000.

Analysis of mRNA Expression by Real Time PCR

Total RNA was isolated from left ventricular tissue and the hypothalamus using TRIzol

reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s specifications. The RNA

concentration was calculated from the absorbance at 260 nm and RNA quality was assured by

260/280 ratio. The RNA samples were treated with DNaseI (Ambion) to remove any genomic

DNA. First strand cDNA was synthesized from 1µg RNA with iScript cDNA synthesis kit

(Bio-Rad, Hercules, CA). Real Time PCR amplification reactions were performed with iTaq

SYBR Green Super mix with ROX (Bio-Rad) in triplicate using the ABI Prism 7900 Real

time PCR machine (Applied Biosystems, Fostercity, CA). The primer sequences were used as

follows: ANP forward 5′-TGC CGG TAG AAG ATG AGG TC-3′; ANP reverse 5′-AGC

CCT CAG TTT GCT TTT CA-3′; BNP forward 5′-AGG GAG AAC ACG GCA TCA TT-3′;

BNP reverse 5′-GAC AGC ACC TTC AGG AGA T-3′; AT1R forward 5′-CTG CGT CTT

GTT CTG AGG TG-3′; AT1R reverse 5′-ACT GGT CCT TTG GTC GTG AG-3′; NF-κB

p50 subunit forward 5′-CGA GGC AGC ACA TAG ATG AA-3′; NF-κB p50 subunit reverse

5′-AGG TCC TTC CTG CCC ATA AC-3′; TNFR1 forward 5′-AAT ATC CTC GAG GCT

CTG AGA-3′; TNFR1 reverse 5′-ATG TAC ACC AAG TTG GTA GC-3′; TNFR2 forward

5′-AGC CCA GGG CGG GAT A-3′; TNFR2 reverse 5′-GGT AAT TCT GGG AAG CCG

3

TAA A-3′. Gene expression was measured by ∆∆CT method and was normalized to 18S

ribosomal RNA or GAPDH mRNA levels. The data are presented as the fold expression of

the gene of interest relative to their control animals.

Statistical Analysis

Results are presented as mean values ± SEM. One-way analysis of variance (ANOVA) was

used for comparisons of results from more than two groups, where as Student t-test was used

to analyze differences between two groups. For repeated measurements analysis a two-way

ANOVA followed by Bonferroni post hoc test was used. GraphPad Prism version 4.03

software (GraphPad Software, San Diego, CA) was used for the analysis. Differences were

considered significant at a value of P < 0.05.

Legend to Supplemental Figure

Figure S1. Effect of angiotensin II infusion on the expression of TNF receptor type 1

(TNFR1) and type 2 (TNFR2) in the left ventricle. A. TNFR1 mRNA expression. B. TNFR2

mRNA expression. C. Representative western blot and densitometric analysis of TNFR1

protein in Left ventricle. Values are mean ± SEM (n=5-6 animals per group). P < 0.05

compared with WT group (*) and TNF-α-/-

group (#).

A

B

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 * #

Fo

ld i

ncre

ase

0.0

0.5

1.0

1.5

2.0

Fo

ld i

ncre

ase

Figure S1:

C

0

1

2

3

4

5

6

*#

WT+Saline

WT+ANG II

TNF--/-

+Saline

TNF--/-

+ANG II

TNF--/-

+ANG II+TNF-

Arb

itra

ry u

nit

s

GAPDH

TNFR1