recombinant human erythopoietin prevents lipopolysaccharide-induced vascular hiporeactivity in the...

7/30/2019 Recombinant Human Erythopoietin Prevents Lipopolysaccharide-Induced Vascular Hiporeactivity in the Rat

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RECOMBINANT HUMAN ERYTHROPOIETIN PREVENTSLIPOPOLYSACCHARIDE-INDUCED VASCULAR HYPOREACTIVITY

IN THE RAT

Roberta dEmmanuele di Villa Bianca,* Rosalinda Sorrentino,* Emma Mitidieri,*Stefania Marzocco, Giuseppina Autore, Christoph Thiemermann,

Aldo Pinto, and Raffaella Sorrentino**Dipartimento di Farmacologia Sperimentale; Universita degli studi di Napoli, Federico II, Napoli;

Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, Salerno, Italy; and William

Harvey Research Institute, Barts and the London School of Medicine and Dentistry, London, UK

Received DD Month YYYY; first review completed DD Month YYYY; accepted in final form DD Month YYYY

ABSTRACTErythropoietin (EPO) is a hypoxia-inducible hormone that is essential for normal erythropoiesis in the bone

marrow. Administration of recombinant humanYEPO is currently being used for the therapy of anemia associated with

chronic renal failure and cancer. Moreover, EPO reduces organ injury in experimental hemorrhagic as well as in splanchnic

artery occlusion shock and preserves cardiac function after experimental cardiac I/R. Erythropoietin receptors are widely

distributed in the cardiovascular system, including endothelial, smooth muscle, cardiac, and other cell types, and

nonhematopoietic effects of EPO are increasingly recognized. Thus, the vasculature may be a biological target of EPO.

Therefore, the aim of our study was to investigate whether EPO exerts a protective effect in septic shock by modulating

vascular dysfunction and hyporeactivity. Rats received EPO(300 U/kg, i.v.) or vehicle 30 minbefore and 1 and 3 h after LPS

(8 106 U/kg, i.v.). In vivo and ex vivo (aortic rings) experiments were performed to evaluate the vascular response to

contracting and vasodilating agents. The expression of iNOS, intercellular adhesion molecule 1, poly(ADP)ribose poly-

merase, Bcl-xl, and Bcl-2 was evaluated by Western blot analysis in the rat aorta. We demonstrate that EPO significantly

prevents LPS-induced vascular hyporeactivity and endothelial dysfunction. Interestingly, EPO inhibited the increase in

iNOS, poly(ADP)ribose polymerase, and intercellular adhesion molecule 1 expression in the aorta of endotoxemic rats and

attenuated the decline in the expression of both Bcl-xl and Bcl-2 caused by LPS. In conclusion, our data support the view

that EPO has important nonerythropoietic effects protecting organ and tissue against injury and indicate that EPO may be

useful in the therapy of patients with septic shock.

KEYWORDSErythropoietin, septic shock, endothelial dysfunction, vasculature injury, rat

INTRODUCTION

Erythropoietin (EPO) is a hypoxia-induced hormone that is

essential for normal erythropoiesis, produced primarily by the

adult kidney. Erythropoietin targets erythroid progenitor cells

in the bone marrow to increase the number of mature red

blood cells (RBCs) (1). The production of recombinant human

(rh)YEPO has revolutionized the treatment of anemia asso-

ciated with chronic renal failure and chemotherapy, and it has

been used as prophylaxis to prevent anemia after surgery. The

EPO receptor is widely distributed in the cardiovascular sys-

tem, that is, located on endothelial cells (2), smooth muscle

cells (3), and cardiomyocytes (4). Although, EPO receptors

are localized on endothelial and smooth muscle cells, EPO

has no direct vasoconstrictor effect in either rabbit aorta orhuman renal artery, but it enhances the contractions caused

by norepinephrine by increasing the synthesis of constrictor

prostanoids and endothelin 1 (5). The probable source of these

vasoconstrictor autacoids is the endothelium, as endothelial

removal attenuates the increase in contraction mediated by

EPO. In endothelial cells from umbilical vein, EPO causes an

increase in prostaglandin F2! and thromboxane B2 and a

decrease in PGI2 (5). At the same time, the release of endo-

thelin 1 is increased by nearly 90% in the presence of EPO (6).

Incubation of bovine pulmonary arterial endothelial cells with

rh-EPO induces a rise of intracellular calcium concentration

accompanied by an increase in endothelin 1 release and in pre-

proendothelin 1 mRNA expression (7). On vascular smooth

muscle cells, EPO causes an increase in angiotensin receptor

messenger RNA, resulting in a parallel increase in the ex-

pression of angiotensin II (Ang II) receptors, which affects va-

somotor tone and remodeling of vascular wall by enhancing

cell proliferation (8). Furthermore, EPO inhibits apoptosis and

induces proliferation and differentiation (9). Taken together,

these findings indicate that EPO is involved in the regulation

of vascular tone through an indirect effect. In addition, EPOreduces the organ injury and dysfunction of rats subjected

to hypovolemic hemorrhagic shock (10); exerts beneficial

effects in splanchnic artery occlusion shock, possibly by in-

hibiting iNOS activity (11); and improves skeletal muscle mic-

rocirculation and tissue bioenergetics in a mouse sepsis model

(12). Moreover, EPO prevents LPS-induced apoptosis in

bovine pulmonary artery endothelial cells, resulting in an in-

crease in viability (13). In addition, EPO protects the myocar-

dial structure and preserves cardiac function during ischemia

and reperfusion (14Y16). The discovery that EPO plays a sig-

nificant biological role in tissues outside the hematopoietic

system has fuelled significant interest in EPO as a novel cyto-

protective cytokine (17). Therefore, the aim of our study was

1

SHOCK, Vol. 00, No. 00, pp. 00Y00, 2008

Address reprint requests to Raffaella Sorrentino, Dipartimento di Farmacologia

Sperimentale, Universita di Napoli Federico II, Via D. Montesano, 49, 80131

Napoli, Italy. E-mail: [email protected].

DOI: 10.1097/SHK.0b013e31818909c0

Copyright 2008 by the Shock Society


2/6Copyright @ 200 by the Shock Society. Unauthorized reproduction of this article is prohibited.8

to investigate whether EPO exerts a protective effect in septic

shock by modulating vascular dysfunction and vascular

hyporeactivity.

MATERIALS AND METHODS

Male Wistar rats weighing 250 to 300 g were used (Harlan, Udine, Italy).Animals were housed in an environment with controlled temperature

(21-

CY

24-

C) and lighting (12:12-h light-dark cycle). Standard chow and drink-ing water were provided ad libitum. A period of 7 days was allowed for ac-climatization of rats before any experimental manipulation was undertaken.Animal use was in accordance with the guidelines of Italian and EuropeanCouncil for animal care. The animals were divided into four experimentalgroups: vehicle plus LPS-treated rats (LPS), EPO plus LPS treated rats(EPO+LPS), control-vehicle (sham), and control-EPO (sham-EPO). Erythro-poietin (300 U/kg; i.v.) or vehicle (saline) was administered 30 min before and 1and 3 h after LPS (8 10

6U/kg, i.v.) or vehicle (saline) injection.

In vivo experimentsHemodynamic changes were evaluated in anesthetized rats by urethane

injection (1 g/kg; i.p.). The trachea was cannulated to facilitate respiration;the right carotid artery was cannulated and connected to a pressure trans-ducer (Bentley 800 Trantec; Basile, Comerio, Italy) for the measurement ofMAP as well as heart rate, which were recorded using the Power Lab/800(AD/Instruments). The left jugular vein was cannulated for administration

of drugs. After surgical procedure, cardiovascular parameters were allowedto stabilize for 20 min. Anesthetized rats received EPO (300 U/kg, i.v.) orvehicle (saline) 30 min before and 1 and 3 h after LPS (8 10

6U/kg, i.v.)

injection. A dose-response effect to phenylephrine (PE, 3, 10, and 30 2g/kg,i.v.) was performed after 4 h from LPS or vehicle (saline) injection. Therecover time between each injection was 15 min.

Ex vivo experimentsIn another set of experiments, rats were treated as described above. At 4 h

after injection of either LPS or vehicle (saline), rats were killed by cervicaldislocation after exposure to isoflurane, and the thoracic aortas were excised,carefully cleaned of connective tissue and cut in rings (2Y3 mm). Rings werehooked in 2.5 mL water-jacketed organ baths filled with thermostated (37-C)and gassed (95% O2 and 5% CO2) Krebs solution with the following com-position (in millimolars): NaCl, 115.3; KCl, 4.9; CaCl2, 1.46; MgSO4, 1.2;KH2PO4, 1.2; NaHCO3, 25.0; and glucose, 11.1. Aortic rings were connec-ted to isometric force transducers (model 7002; Basile), and changes in ten-

sion were recorded continuously using a polygraph linearcorder (WR3310;Graphtec, Japan). Tissues were preloaded with 0.5 g and allowed equilibratingfor at least 90 min; during this time, Krebs solution was changed about each15 min. After equilibration, the tissues were used to evaluate vascular re-

activity. The contractile response was evaluated by using PE (1 nM up to 3 2M),h-endothelin 1(hYEt-1, 30 nM), U46619, a stable analog of thromboxane A2(0.3 2M) and Ang I (0.1 2M). To assess the endothelial function, we per-formed a concentration-response curve with acetylcholine (ACh, 10 nM to3 2M) in aortic rings precontracted by PE (0.3 2M). In another set of ex-periments to evaluate the release of NO, we estimated the increase in L-NAME(100 2M)Yinduced contraction added on stable tone of PE (0.3 2M).

Western blot analysis for iNOS, intercellular adhesionmolecule 1, poly(ADP)ribose polymerase, Bcl-xl,and Bcl-2 expression

Briefly, frozen arteries were homogenized in a lysis buffer (50 mM"-glycerophosphate, 100 2M Na3VO4, 2 mM MgCl2, 1 mM EGTA, 0.5%triton, and 1 mM DTT) containing protease inhibitors (20 2M pepstatin,20 2M leupeptin, 1,000 U/mL aprotinin and 1 mM PMSF). Protein concentra-tion was estimated by Bio-Rad protein assay using bovine serum albumin asstandard. Equal amounts of protein (50 2g) were dissolved in Laemmlisample buffer, boiled and run on a sodium dodecyl sulfateYpolyacrylamide gelelectrophoresis minigel, and then transferred to a Hybond polyvinylidene dif-luoride membrane. Membranes were then blocked for 40 min in PBS contain-ing 5% (wt/vol) nonfat milk and subsequently probed overnight at 4-C withmouse monoclonal anti-iNOS or antiYintercellular adhesion molecule 1 (ICAM-1) or antiYpoly(ADP)ribose polymerase (PARP) or antiYBcl-xl or antiYBcl-2(1:2,500, 1:600, 1:400, 1:750, and 1:750 dilution, respectively, in PBS contain-ing 5% wt/vol nonfat milk and 0.1% Tween-20). Blots were then incubated, after4 washes in PBS containing 5% wt/vol nonfat milk and 1% Tween-20, withhorseradish peroxidaseYconjugated goat antiYmouse IgG (1:5,000 for iNOS,Bcl-xl, and Bcl-2 detection) or with horseradish peroxidaseYconjugatedgoatYanti-rabbit IgG (1:5,000 for ICAM-1 and PARP detection) for 1 h atroom temperature. Immunoreactive bands were visualized using ECL detec-tion system according to the manufacturers instructions and exposed to Kodak

X-Omat film. Protein bands on x-ray film were quantified by scanning densi-tometry (Imaging Densitometer GS-700; Bio-Rad) and results normalized withtubulin tissue expression, as reference protein. Densitometric analyses wereperformed using the NIH Image program.

Evaluation of RBCs, Hb, and hematocritBlood samples were withdrawn before and after each treatment as

described above to evaluate changes in hematocrit (HCT), RBCs, and he-moglobin (Hb) using a cell counter (Coulter AcT Diff 2; InstrumentationLaboratory, Italy).

Statistical analysisAll values in the figures, table, and text are expressed as mean T SEM.

The statistical analysis was performed using GraphPad Prism (GraphPadSoftware, San Diego, Calif) by one or two-way ANOVA followed byBonferroni post hoc test. A P G 0.05 was taken as significant. The increasein MAP induced by PE was expressed as delta compared with the basal value.

The contraction afforded by a given drug ex vivo was expressed as dyne per

FIG. 1. Effect of EPO on LPS-induced hypotension in anesthetizedrats. Blood pressure values are reported as MAP and expressed inmillimeters of mercury. The MAP was monitored for 4 h after LPS challenge(time 0). Insert, Early phase (0Y30 min). Anesthetized rats received EPO(300 U/kg, i.v.) or vehicle (saline) 30 min before and 1 and 3 h after LPS (8

106 U/kg, i.v.) injection. Results are expressed as mean T SEM for LPS (n =10), LPS+EPO (n = 7), sham-EPO (n = 6), and sham (n = 5) groups. Dataare analyzed by two-way ANOVA followed by Bonferroni as posttest. P G

0.05 was taken as significant. ###P G 0.0001 vs. LPS; **P G 0.005 LPS or

LPS+EPO vs. sham.

FIG. 2. Effect of EPO on LPS-induced hyporeactivity to PE (3, 10, and30 2g/kg, i.v.) after 4 h from LPS or vehicle injection . Anesthetized ratsreceived EPO (300 U/kg, i.v.) or vehicle (saline) 30 min before and 1 and 3 hafter LPS (8 106 U/kg, i.v.) injection. Results are expressed as delta ofincrease in MAP of mean T SEM of LPS (n = 10), LPS+EPO (n = 7), sham-EPO (n = 6), and sham (n = 5) groups. Data are analyzed by one-wayANOVA followed by Bonferroni as posttest. P G 0.05 was taken assignificant. *P G 0.005 and **P G 0.01 vs. sham; -P G 0.05 and --P G 0.01

vs. LPS.

2 SHOCK VOL. 00, NO. 00 DI VILLA BIANCA ET AL.



milligram of tissue. Any relaxation caused by a drug in the same ex vivoexperiments was expressed as percentage of relaxation.

RESULTS

In vivo study

The baseline values for MAP were not significantly differ-

ent between any of the groups studied. The MAP values were

100T

3 (n = 10), 109T

7 (n = 7), 105T

3 (n = 6), and 102T

4 mmHg (n = 5) for LPS, LPS+EPO, sham-EPO, and sham,

respectively. When compared with sham, LPS administration

caused a significant hypotension (P G 0.001; Fig. 1). In-

terestingly, EPO attenuated the hypotension observed in

LPS-treated rats (P G 0.001; Fig. 1). The MAP values of

sham rats treated with EPO (sham-EPO) were not different

FIG. 3. Effect of EPO on vascular reactivity to different pressor agents in ex vivo experiments in rat aorta rings. The contractile response wasevaluated after 4 h from treatments EPO (300 U/kg, i.v.) or vehicle (saline) 30 min before and 1 and 3 h after LPS (8 106 U/kg, i.v.) injection. A, PE (1 nmol upto 3 2M); **PG 0.001 vs. LPS and sham-EPO, ---PG 0.0001 vs. sham-EPO and LPS. B, Ang II (Ang, 0.1 2M); *PG 0.05 vs. LPS, **PG 0.01 vs. LPS, ***PG

0.001 vs. LPS, -PG 0.05 vs. EPO+LPS, and --PG 0.01 vs. EPO+LPS. C, U46619, a stable analog of thromboxane A2 (0.3 2M); *PG 0.05 vs. EPO+LPS andsham-EPO. D, hYEt-1(h-ET-1, 30 nM); *PG 0.05 vs. EPO+LPS, **PG 0.005 vs. LPS. A cumulative concentration curve was performed with PE, whereas asingle concentration response was used for Ang, U46691, and hET-1. Results are expressed in dyne/mg of tissue as mean T SEM of LPS (n = 8), EPO+LPS(n = 8), sham-EPO (n = 5), and sham (n = 5) groups. Data are analyzed by two-way (A) and one-way (B, C, and D) ANOVA , followed by Bonferroni asposttest. PG 0.05 was taken as significant.

FIG. 4. Effect of EPO on endothelial dysfunction caused by LPS (8 106 U/kg, i.v.). A, ACh (10 nmol to 3 2M) concentration-response curve in aortarings precontracted by PE (0.3 2M); ***PG 0.001 vs. EPO+LPS and sham, **PG 0.01 vs. LPS. Results are expressed as % of relaxation to PE tone. B, L-NAME(100 2M)Yinduced contraction added on stable tone of PE (0.3 2M); *PG 0.05 vs. LPS; **PG 0.01 vs. LPS. Results are expressed as % of delta increase to PEtone. Data are expressed of mean T SEM of LPS (n = 12), EPO+LPS (n = 12), sham-EPO (n = 5), and sham (n = 5) groups and analyzed by two-way (A) and

one-way (B) ANOVA, followed by Bonferroni as posttest. PG 0.05 was taken as significant.

SHOCK MONTH 2008 EPO PREVENTS LPS-INDUCED VASCULAR INJURY 3



from those measured in sham rat. The vascular reactivity

in vivo to PE (3, 10, and 30 2g/kg i.v.) was evaluated 4 h after

LPS or vehicle injection. Compared with the sham group, asignificant hyporeactivity to PE was observed in the LPS

group (3 and 10 2g/kg, P G 0.01; 30 2g/kg, P G 0.05; Fig. 2).

Erythropoietin treatment completely reverted the vascular

hyporeactivity to PE induced by LPS (3 2g/kg, P G 0.05; 10

and 30 2g/kg, P G 0.005; Fig. 2); indeed, LPS+EPO rats

showed a reactivity to PE that was statistically indistinguish-

able to sham group. In the sham-EPO group, the PE-induced

variation in blood pressure was similar to those produced in

the sham and LPS+EPO groups (Fig. 2).

Ex vivo study

Vasoconstrictor responsesAt 4 h after administration of

LPS, rats were killed, and aortic rings were suspended inorgan baths and challenged with different contracting agents.

The contraction of aortic rings caused by PE (3nM to 3 2M)

was significantly reduced in ex vivo LPS-treated rats compared

with the sham group (P G 0.0001; Fig. 3A). Erythropoietin

treatment (LPS+EPO) attenuated the LPS-induced hypo-

reactivity to PE (P G 0.0001; Fig. 3A). In aortic rings collected

from sham-EPO group, the concentration-response curve to

PE was significantly increased compared with the sham group

(P G 0.0001; Fig. 3A). In the same way, aortic rings of LPS

rats showed a significant (P G 0.05) reduced response to Ang(0.1 2M), U-46619 (0.3 2M), or hYEt-1 (30 nM) compared

with the sham group (Fig. 3, BYD). Similarly to response to

PE, aortic rings of LPS+EPO rats exhibited a contraction to

Ang (0.1 2M), U-46619 (0.3 2M), or hYEt-1 (30 nM) com-

pletely comparable to sham group (Fig. 3, BYD). Erythro-

poietin treatment did not affect the response to Ang, U-46619,

or hYEt-1 in sham animals.Acetylcholine-induced relaxation and NO releaseTo

assess the integrity of the endothelium, an ACh-induced

concentration-response curve was performed. In endotoxemic

rats, ACh-induced relaxation was significantly reduced (P G

0.0001) compared with the sham group, indicating the devel-

opment of an endothelial dysfunction, which was prevented byEPO treatment (LPS+EPO group; Fig. 4A). Indeed, ACh-

induced relaxation in LPS+EPO aortas was similar to that

observed in the sham group (Fig. 4A). Erythropoietin treatment

did not modify the relaxation induced by ACh in sham animals

(sham-EPO; Fig. 4A). The increase in contracting force of

aorta rings, displaced by adding L-NAME (100 2M), an in-

hibitor of NOS, on the stable tone of PE-induced contraction

FIG. 5. Effect of EPO on the expression of different protein in rat aorta after 4 h from treatments by Western blot analysis. A, iNOS expression(##PG 0.01 vs. sham, **PG 0.01 vs. LPS). B, ICAM-1 expression (###PG 0.001 vs. sham, *PG 0.0 vs. LPS). C, Cleaved PARP fragment expression (##PG 0.01vs. sham, **PG 0.01 vs. LPS). Protein bands were quantified by scanning densitometry, and results are reported as integrated values (area density of the

band). All values are expressed as means T SEM of three different experiments with three replicates in each. PG 0.05 was taken as significant.

FIG. 6. Effect of EPO on the expression of different proteins in rat aorta after 4 h from treatments by Western blot analysis . A, Bcl-2 expression(###PG 0.001 vs. sham, **P G 0.01 vs. LPS. B, Bcl-xl expression ((###P G 0.001 vs. sham, *P G 0.05 vs. LPS). Protein bands were quantified by scanningdensitometry, and results are reported as integrated values (area density of the band). All values are expressed as means T SEM of three different

experiments with three replicates in each. PG 0.05 was taken as significant.




(0.3 2M), was assumed as an index of NO release. Aortas of

LPS-treated rats showed a significant (P G 0.01) increase in NO

release than in aortas obtained from the sham group. In

LPS+EPO rats, the NO release was significantly (P G 0.05) re-

duced compared with the LPS group (Fig. 4B) and not statisti-

cally different from the sham-EPO and sham groups (Fig. 4B).Western blot studyWestern blot analysis revealed a sig-

nificant (PG

0.005) increase in iNOS protein expression inaorta of LPS group compared with sham, LPS+EPO, and

sham+EPO groups (Fig. 5A). The expression of iNOS pro-

teins resulted significantly attenuated in LPS+EPOYtreated

rats; whereas no difference was observed in sham+EPO and

LPS+EPO compared with the sham group. Endotoxemia

(LPS group) also resulted in an increased of ICAM-1 and

PARP cleaved form (89 kd) protein expression (P G 0.01;

Fig. 5, B and C, respectively), and both of these effects were

significantly (P G 0.05) attenuated by EPO treatment. Bcl-2

and Bcl-xl protein expressions, which inhibit both apoptosis

and proliferation, were significantly reduced in the aorta of

LPS-treated rats (P G 0.01; Fig. 6, A and B). Interestingly,

EPO treatment significantly attenuated the decline in the ex-pression of both Bcl-2 and Bcl-xl caused by LPS (P G 0.05;

Fig. 6, A and B).

Evaluation of RBCs, HCT, and HbRed blood cell count,

Hb, and HCT (%) were measured in all rats before each

treatment (basal) and 4 h after challenge with LPS or vehicle.

Red blood cell, Hb, and HCT values were not changed in each

group (Table 1).

DISCUSSION

The presence of EPO receptors on several cell types, that

is, neurons, cardiomyocytes, endothelial, vascular smooth

muscle, and kidney cells such as proximal tubule epithelialand mesangial cells and the glomerulus (2Y4, 18, 19), indi-

cates that EPO, as a growth factor or cytokine, may have

effects on many cells and tissues. We demonstrated here that

EPO attenuates both hypotension and vascular hyporeactivity

in an in vivo and ex vivo model of endotoxemic shock in

rat. Similar beneficial effects have been reported for EPO in

other animal models of shock, including hemorrhagic and

splanchnic artery occlusion shock (10, 11). We found that

EPO prevented early hypotension (30 min) and, moreover,

the long-lasting hypotension induced by LPS administration.

The response to the !1 receptor agonist PE was significantly

reduced, indicating the development of a vascular hyporeac-

tivity in endotoxemic rats. Most notably, EPO significantly

improved the PE response in LPS-treated rats in vivo. Our

in vivo data correlated with our ex vivo results obtained in the

isolated aorta. Specifically, EPO pretreatment prevented LPS-

induced hyporeactivity to PE in isolated aortic rings. Surpris-

ingly, EPO significantly augmented the response to PE in

aortic rings not subjected to endotoxemia, while such an effect

was not observed in vivo (above). The latter result may be due

to effects of anesthesia and/or by blood pressure autoregula-tion. The increase observed in !1 response induced by EPO

ex vivo is in accord with the studies of Bode-Boger and col-

leagues (5, 6). Indeed, they have demonstrated that the en-

hanced contractile response to noradrenalin involves a shift in

the balance of constrictor and relaxing prostanoids (5, 6). This

imbalance in prostanoid production seems to be closely re-

lated to !1 adrenergic receptor pathway. Thus, we propose

that the increase in the response to PE afforded by EPO,

which was not observed when we used thromboxane A2, en-

dothelin 1, or Ang as vasoconstrictor, may be secondary to the

release by EPO of endogenous vasoconstrictor autacoids

(5Y8). However, EPO also attenuated the LPS-induced

vascular hyporeactivity to the vasoconstrictor responses eli-cited by h-endothelin, thromboxane A2, and Ang II. More-

over, the improvement in vascular hyporeactivity induced by

LPS may be related to an increase in intracellular calcium

concentration induced by EPO (20). Interestingly, EPO at-

tenuated the impairment in ACh vasodilator response caused

by LPS, indicating that this hormone also prevented the endo-

thelial dysfunction caused by LPS. In fact, it has been demon-

strated that EPO attenuates the cardiac injury and dysfunction

caused by regional myocardial ischemia in the rat and rabbit

(15, 21, 22). This beneficial effect of EPO has been ascribed

to preservation of endothelial function and vascular flow in

coronary vessel secondary to antiapoptotic effects of EPO.Our molecular data are in this direction; in fact, we found

that the impaired expression of the antiapoptotic proteins

Bcl-2 and Bcl-xl in aortas of LPS group was significantly

attenuated by EPO treatment. All of the above data support

the hypothesis that EPO prevents the endothelial dysfunction

and injury caused by LPS. Noteworthy are the results obtained

for PARP, a nuclear enzyme activated by DNA damage. Dur-

ing DNA damage, PARP is activated as a DNA-repair en-

zyme by formation of poly(ADP-ribose) in a process that

consumes ATP. The excessive DNA damage associated with

a huge activation of PARP leads to a cascade of events, known

as the Bsuicide pathway[ (23) that causes necrosis (24) and

apoptosis (25). Poly(ADP)ribose polymerase inhibitors were

TABLE 1.

Treatment

Basal 4 h

Hb, g/dL RBCs, 106/2L HCT, % Hb, g/dL RBCs, 106/2L HCT, %

Sham 14.10 T 0.2 7.85 T 1.4 48.30 T 2.1 14.33 T 2.3 10.77 T 2.7 52.00 T 1.2

LPS 14.38 T 0.4 9.10 T 0.3 51.12 T 1.7 14.23 T 0.4 9.50 T 0.3 52.88 T 1.5

Epo+LPS 14.02 T 0.7 8.54 T 0.4 47.08 T 2.0 14.18 T 1.1 9.46 T 0.9 53.48 T 5.0

Sham-EPO 14.68 T 0.6 9.68 T 0.4 50.38 T 0.8 13.90 T 1.0 9.91 T 0.8 52.58 T 3.0

Red blood cell count and HCT were measured in rats at different points: basal (at the beginning of the experiment) and 4 h after LPS challenge. Hb,RBC count, and HCT were not changed in LPS rats. In addition, EPO treatment did not modify Hb, RBC count, and HCT either in sham or LPS+EPOgroup. The values are reported as mean T SEM of sham (n = 5), LPS (n = 10), LPS+EPO (n = 7), and sham-EPO (n = 6) groups.

SHOCK MONTH 2008 EPO PREVENTS LPS-INDUCED VASCULAR INJURY 5


6/6C i ht @ 200 b th Sh k S i t U th i d d ti f thi ti l i hibit d8

found to be beneficial in many pathophysiological condi-

tions associated with oxidative stress and, moreover, PARP

knockout mice proved to be resistant to LPS-induced shock

(26). Our data showed an increase in the formation of PARP

in aortas obtained from rats subjected to endotoxemia that was

completely reverted by EPO administration. This result, in

addition to the observed effect on Bcl-2 and Bcl-xl, indicates

that EPO may prevent LPS-induced apoptosis in aortas. A

further enzyme involved in cell damage is iNOS, which ex-

pression in many cell types, including macrophages and

vascular smooth muscle cells, is triggered by LPS (27). The

overproduction of NO by iNOS could be responsible in part

for the endothelial and DNA damage, because simultaneous

production of NO and superoxide anions in LPS-treated ani-

mals leads to the production of peroxynitrite and subsequently

nitrotyrosine formation in the blood vessels (28). Erythro-

poietin contributed to the protective effect of vascular wall by

inhibition of iNOS expression. Furthermore, the reduction in

iNOS expression was accompanied by significant diminution

in NO formation, as demonstrated by L-NAMEY

induced con-traction, which could be involved in the improvement of

vascular hyporeactivity. Similarly, it has been reported that

EPO protects against splanchnic artery occlusion shock

by inhibiting the expression of iNOS protein (11). A further

index of LPS-induced cellular damage is ICAM-1 expres-

sion. Erythropoietin also attenuated the expression of the ad-

hesion molecule ICAM-1, resulting in a reduced adhesion of

neutrophils to the endothelium. It should be noted that the

observed beneficial effects of EPO are not related to any im-

provement in HCT or Hb, as the dose of EPO used had no

effect on these parameters. Our data support the hypothesis

that EPO has important nonerythropoietic effects, which may

protect tissues and organs against injury. Taken together, ourdata support the view that a low dose of rh-EPO exerts bene-

ficial effects in rodent models of endotoxemia. As sustaining

blood pressure is critical for the survival of humans with se-

vere septic shock (29), we speculate that our data indicate that

EPO may be useful in the therapy of patients with septic

shock, but clearly, further studies are warranted to support this

working hypothesis.

ACKNOWLEDGMENTS

The authors thank medical veterinarians Dr Lucia DEsposito, Giovanni

Esposito, and Angelo Russo for animal care assistance.

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