Andrenomedullin and cardiovascular responses in sepsis

Peptides 22 (2001) 1835–1840

Andrenomedullin and cardiovascular responses in sepsis Ping Wang* Center for Surgical Research and Department of Surgery, School of Medicine, University of Alabama at Birmingham, Volker Hall, Room G094P, 1670 University Boulevard, Birmingham, AL 35294, USA Received 05 March 2001; revised 12 April 2001; accepted 15 August 2001

Abstract The typical cardiovascular response to polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Although the factors and/or mediators responsible for producing the transition from the hyperdynamic to the hypodynamic stage are not fully understood, recent studies have suggested that adrenomedullin (AM), a potent vasodilatory peptide, appears to play an important role in initiating the hyperdynamic response following the onset of sepsis. In addition, the reduced vascular responsiveness to AM may result in the transition from the early, hyperdynamic phase to the late, hypodynamic phase of sepsis. It is possible that changes in newly reported AM receptors calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-2 or -3 (RAMP2, RAMP3) as well as AM binding protein-1 (AMBP-1) may also play distinct roles in the biphasic cardiovascular response observed during sepsis. Although it remains unknown whether AM gene delivery or a chronic increase in vascular AM production in transgenic animals attenuates the development of hypodynamic sepsis and septic shock, it has been shown that modulation of AM vascular responsiveness with pharmacologic agents reduces sepsis-induced mortality. It has been recently demonstrated that AMBP-1 enhances AM⬘s physiologic effects and plasma levels of AMBP-1 decrease following infections. We therefore propose that downregulation of AMBP-1 and the reduced AM receptor responsiveness are crucial factors responsible for the transition from the hyperdynamic phase to the hypodynamic phase of sepsis. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Hemodynamic responses; Vascular responsiveness; Proinflammatory cytokines; Adrenomedullin receptors; Hyperdynamic sepsis; Septic shock; Cecal ligation and puncture

1. Introduction Sepsis, septic shock, and multiple organ failure continue to be the major causes of mortality in intensive care units [2,5,13]. The cardiovascular response to polymicrobial sepsis in an animal model [i.e. cecal ligation and puncture (CLP)] is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase [61,68]. The CLP model of sepsis is analogous to perforated appendicitis and mimics many features of clinical peritonitis [6,62,67]. The CLP model produces a hemodynamic response which consists of an early, hyperdynamic phase (characterized by increased cardiac output and tissue perfusion, decreased vascular resistance, hyperglycemia, and hyperinsulemia from 2–10 h after CLP), followed by a late, hypodynamic phase (characterized by decreased tissue perfusion, hypoglycemia, and hypoinsulemia 16 h after CLP and later) * Corresponding author. Tel.: ⫹1-205-975-9714; fax: ⫹1-205-9759715 E-mail address: [email protected] (P. Wang).

[61,66,68]. This model therefore allows the assessment of animals during both the hyperdynamic and hypodynamic phases of sepsis. Moreover, by using the CLP model of sepsis, mediators/factors which are responsible for the transition from hyperdynamic sepsis to hypodynamic sepsis, can be examined. Recent studies have suggested that the potent vasodilatory peptide adrenomedullin (AM) appears to play an important role in producing the hyperdynamic response as well as the transition to the hypodynamic phase of sepsis [28,55,60,63,64]. AM was first isolated by Kitamura et al. from human pheochromocytomas and reported in 1993 [25]. This peptide has a 6-residue structure formed by an intramolecular disulfide-bridge and a carboxy-terminal amidated residue. The complementary DNA encoding human or rat AM has been cloned and sequenced [26,44]. Rat AM has 50 amino acid residues, with two amino acid deletions and six substitutions compared with human AM [44]. AM transcripts and proteins are expressed in a large number of tissues and cell populations [1,3,26,33,44]. AM’s actions are associated with cardiovascular, endocrine, and renal mechanisms that

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control fluid and electrolyte homeostasis [45]. The vasodilatory properties of AM are of particular interest in the pathophysiology of sepsis. It has been suggested that AM elicits its vasodilatory effect through two different mechanisms: a direct effect on vascular smooth muscle cells to increase intracellular cAMP by stimulating AM receptors and adenylate cyclase [8,9,32], and an indirect effect on vascular endothelial cells by increasing endothelium-derived nitric oxide (NO) release [46]. Administration of AM in conscious sheep decreases blood pressure and increases heart rate, which is associated with an increased cardiac output and coronary blood flow and decreased peripheral vascular resistance [42]. Similarly, administration of AM in the rat increases cardiac output and blood flow in the heart, lungs, kidneys and small intestine and decreases blood pressure and vascular resistance [16]. In addition, we have demonstrated that administration of synthetic rat AM at a dose similar to that observed during early sepsis, which does not produce significant alterations in blood pressure or heart rate, markedly increases cardiac output, stroke volume, and tissue perfusion while decreasing total peripheral resistance [60]. This would suggest that upregulation of AM observed during sepsis may play an important role in producing the early, hyperdynamic response [55].

2. Upregulation of AM in sepsis It has been shown that circulating levels of AM increase significantly during sepsis either in patients [17] or in experimental animals [64]. Upregulation of AM was also found under pathophysiologic conditions which share some common characteristics with sepsis and septic shock, such as systemic inflammatory response syndrome [54], hemorrhagic shock and endotoxic shock [14,24,37], and hypoxia [4,19,35]. Endotoxin has been proposed as a leading mediator responsible for stimulating AM production during sepsis [65]. In this regard, the upregulation of AM can also be observed after administration of endotoxin in vivo as well as in vitro [49,52]. AM gene expression in endotoxin-injected animals is augmented in the small intestine, liver, lungs, aorta, heart and skeletal muscle [47,49,50]. In addition, the small intestine shows the highest increment ratio of AM mRNA after endotoxin administration [49], suggesting that the upregulated of AM in the small intestine may contribute to the increased portal blood flow observed during sepsis [56]. This finding is further supported by studies from our laboratory, indicating that the small intestine is indeed a major AM-producing organ during polymicrobial sepsis [72]. AM immunoreaction products were primarily located in connective tissues of the lamina propria and submucosa. In addition, AM immunoreaction products were also observed in the intestinal nerve fibers which surround the intestinal glands and small blood vessels. To the best of our knowledge, this is the first report indicating that AM-positive immunostainings are localized in the intestinal nerve

fibers during polymicrobial sepsis. Thus, connective tissues of the lamina propria and submucosa and intestinal nerve fibers are responsible for AM production in the small intestine during sepsis [72]. To determine the specific role of endotoxin in stimulating AM production, we have recently conducted the following experiment [65]. 200-␮l Alzet micro-osmotic pumps, containing 0.375 ␮g E. coli lipopolysaccharide (LPS, 055:B5, Difco) or vehicle (saline) were implanted in the peritoneal cavities of normal male adult rats. At 10 h after pump implantation (which produced plasma levels of LPS similar to that seen during sepsis, as measured by the limulus amebocyte lysate assay), samples of blood and small intestine were harvested for the determination of AM by radioimmunoassay. In additional groups, rats were subjected to sepsis by CLP. LPS binding agent polymyxin B (PMB, 600 U/kg body wt., which markedly decreased plasma levels of LPS) was administrated intramuscularly (i.m.) at 1 h prior to and at 5 h after CLP. At 10 h after CLP or sham-operation, blood and intestinal levels of AM were determined. The results indicate that continuous administration of a low dose of LPS in normal animals (which did not alter cardiac output, blood pressure, or heart rate) significantly increased AM production. Conversely, the LPS binding agent PMB attenuated the elevated AM in plasma and intestinal tissues during sepsis. We therefore concluded that the upregulated AM observed during polymicrobial sepsis is at least in part due to the increase in circulating levels of LPS [65]. Proinflammatory cytokines such as TNF-␣ and IL-1␤ may also participate in the process of AM upregulation during sepsis. Previous studies have indicated that circulating levels of TNF-␣ and IL-1␤ increase significantly in sepsis [11]. Additionally, it has been demonstrated that circulating levels of TNF-␣ and its mRNA in Kupffer cells increase even earlier than the onset of hyperdynamic circulation during sepsis [57,58]. Moreover, IL-1␤ gene expression increases early after the onset of sepsis [31]. In this regard, AM gene expression was found to be markedly augmented by administration of TNF-␣ [53]. Similarly, AM secretion and its mRNA levels in cardiac myocytes are increased after stimulation with IL-1␤ [20]. It has been shown that endotoxin and proinflammatory cytokines such as TNF-␣ and IL-1␤ synergistically enhance the production of AM in cultured vascular smooth muscle cells [52]. Since the above-mentioned mediators increase earlier than the increase in circulating levels of AM [64], it appears that endotoxin and/or proinflammatory cytokines are responsible for upregulating AM production after the onset of sepsis.

3. The role of AM in the cardiovascular responses in sepsis Previous studies have demonstrated that the biphasic hemodynamic response occurs during the progression of polymicrobial sepsis [61,62,68]. The early, hyperdynamic

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response to sepsis (i.e. 2–10 h after CLP) is characterized by an increase in cardiac output and regional perfusion, oxygen delivery and consumption, and a decrease in systemic vascular resistance. In contrast, the late, hypodynamic response (16 h after CLP or later) is characterized by a decrease in cardiac output, blood flow and oxygen delivery, and an increase in vascular resistance. It has been shown that plasma levels of AM increase as early as 2 h after CLP, progressively increase from 5–20 h after, and remain elevated at 30 h after the onset of sepsis [64]. Similarly, circulating levels of AM also increase significantly in patients with sepsis and septic shock [17,36]. The increase in circulating levels of AM is associated with upregulated gene expression of this peptide in the small intestine, left ventricle, and thoracic aorta but not in renal and hepatic tissues after CLP [64]. It appears that the gut is a major source of AM release during sepsis as evidenced by higher levels of AM in portal than in systemic blood [72]. Moreover, AM immunostainings increase in cardiovascular tissues (mainly in vascular endothelial cells and cardiac myocytes) during sepsis [71]. The lungs appear to be the primary site of AM clearance [18], which is significantly diminished during the late stage of sepsis [39]. Since upregulation of AM expression and increase in its plasma levels occur coincidently with the hyperdynamic response during the early stage of sepsis, it is possible that there is a cause and effect relationship between the two events. This hypothesis was confirmed by a study which demonstrated that i.v. infusion of synthetic rat AM, which increased plasma levels of AM similar to those observed during sepsis, produced the characteristic hyperdynamic response, including increased cardiac output, microvascular blood flow, and decreased total peripheral resistance [60]. Similarly, administration of AM in healthy human volunteers increases cardiac output and lowers diastolic pressure [29]. In addition, the administration of anti-AM antibodies at 1.5 h after CLP, which completely neutralized circulating AM, prevented the hyperdynamic response [60]. Thus, the increased AM production and release plays a major role in producing the hyperdynamic response during early sepsis. To determine the factors responsible for producing the transition from the hyperdynamic to the hypodynamic phase of sepsis, studies were performed to examine vascular responsiveness to AM stimulation during sepsis. The results indicated that despite upregulated levels of AM even in late sepsis, vascular responsiveness to AM decreases at both the macro- and microcirculatory levels [63]. It is possible that the reduction in vascular responsiveness to AM leads to a deterioration in hemodynamics and is responsible for producing the transition from the hyperdynamic to the hypodynamic stage of sepsis. Although the precise mechanism responsible for the reduction in vascular responsiveness to AM remains unknown, it is likely that alterations occur in membrane-bound AM receptors and/or the related signal transduction during the progression of sepsis. In this regard, it has been reported that AM per se can produce desensiti-

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zation of adenylate cyclase coupled to vascular AM receptors [22]. Other plausible explanations of decreased vascular AM responsiveness during the late stage of sepsis include the internalization of AM receptors, downregulation of AM binding sites, or a decrease in AM receptor gene expression. In addition, a decrease in AM binding proteins [10] may also play a role in altering the bioactivity of AM during the late stage of sepsis. Although AM is a circulating hormone, it has been suggested that this peptide also acts as a local autocrine and/or paracrine vasoactive hormone [23]. This suggestion has been made in view of the studies which showed that AM, if injected i.v., increases flow rate predominantly in organs in which the AM gene is highly expressed [23].

4. AM receptors and AM binding proteins in sepsis Using radioactively labeled AM, investigators have shown specific binding sites on a large number of tissues such as the intestine, heart, lungs, spleen, liver, soleus, diaphragm, and spinal cord membranes [41]. It has been recently demonstrated that AM receptors and CGRP receptors have a close relationship which is dependent upon the receptor activity modifying proteins (RAMPs) which transport the receptors to the plasma membrane [30]. It has been identified that AM receptor components are calcitonin receptor-like receptor (CRLR), RAMP2 and RAMP3 [30]. In this regard, studies by Ono et al. indicate that while pulmonary expression of CRLR and RAMP2 mRNAs was markedly reduced following endotoxemia (2 mg/mouse), RAMP 3 gene expression increased in the lungs, spleen, and thymus under such conditions [38]. Similarly, our preliminary data indicate that RAMP3 mRNA in lungs increased significantly during the early, hyperdynamic stage, but not during the late, hypodynamic stage of sepsis [40]. In contrast to the work reported by Ono et al. in a mouse model of endotoxemia [38], we did not observe significant changes in pulmonary CRLR and RAMP2 gene expression in a rat model of polymicrobial sepsis. Nonetheless, it remains unknown whether the difference in AM receptor expression in those studies is due to the difference in the nature of pathophysiologic conditions or due to different species. It also remains unknown whether the reduced vascular responsiveness to AM stimulation observed during sepsis [63] is due to reduction of AM receptor gene expression, alteration in receptor affinity, or receptor-adenylate cyclase uncoupling. Recent studies by Elsasser et al. [10] have demonstrated the presence of specific AM binding proteins (120 and/or 140 kDa) in mammalian blood. Pı´o et al. further characterized the specific AM binding protein (AMBP-1) and purified AMBP-1 to homogeneity [43]. The purified human AMBP-1 is identical to human complement factor H. Since the routine procedure of radioimmunoassay does not take into account the AM bound to AMBP-1, it is likely that the plasma levels of AM reported in the literature only repre-

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sents the levels of free AM [43]. More importantly, the authors reported that AMBP-1 potentiates AM-induced cAMP accumulation in cultured Rat-2 cells [43], suggesting that AMBP-1 may play an important role in AM-induced vascular relaxation. Thus, the specific binding protein for AM (AMBP-1) in the circulation may affect the bioactivity and function of AM under normal as well as disease conditions. Although it remains unknown whether circulating levels of AMBP-1 are altered during polymicrobial sepsis, studies have shown that there was a 67% decrease in AM binding protein in the plasma of calves undergoing an acute phase response to a parasitic infection compared to healthy calves [10].

5. Modulation of AM in sepsis and future area of research Recent studies have indicated that AM suppresses IL1␤-induced TNF-␣ production in Swiss 3T3 cells [21]. This would suggest that AM may also be an anti-inflammatory mediator. Similarly, studies by Yoshibayashi et al. suggest that intrinsically increased AM levels may function as a compensatory mechanism for hypoxemia in congenital cyanotic heart disease [70]. Due to the fact that vascular responsiveness to AM and AMBP-1 decrease following infection [10,63], we hypothesize that the lack of adequate production of AM following the onset of sepsis plays an important role for the development of the hypodynamic and hypocardiovascular response. We further hypothesize that enhancement of early production of AM prolongs the hyperdynamic and hypercardiovascular response and thereby reduces sepsis-induced multiple organ failure and late mortality. By using transgenic mice overexpressing AM in their vasculature, Shindo et al. recently reported that administration of bacterial LPS elicited a smaller decline in blood pressure and less severe organ damage in AM transgenic mice than in wild-type mice [48]. Furthermore, the 24-h survival rate after induction of endotoxic shock was significantly higher in the transgenic mice, suggesting that AM is protective against the circulatory collapse, organ damage, and mortality characteristic of endotoxic shock [48]. Although the AM gene delivery technique has not been utilized in sepsis or endotoxic shock, Dobrzynski et al. have nicely demonstrated that somatic human AM gene delivery attenuated hypertension, protected against cardiac remodeling and renal damage in an animal model of volume-overload hypertension, and may have significance in therapeutic application in cardiovascular and renal diseases [7]. Since deterioration in vascular reactivity may lead to hemodynamic instability and be responsible for the transition from the hyperdynamic to the hypodynamic phase [15], maintenance of vascular responsiveness to various vasoactive agents becomes very important in sepsis. In order to determine whether pharmacologic agents would modulate vascular responsiveness to AM stimulation, studies have been conducted to examine the effects of the phosphodies-

terase inhibitor pentoxifylline (PTX) on vascular reactivity in response to AM stimulation during sepsis [27]. PTX has been extensively studied during sepsis and has been shown to produce a number of beneficial effects [51,59]. It has been recently demonstrated that early administration of PTX maintains hemodynamic stability and oxygen utilization in the late stage of sepsis in addition to reducing mortality [69]. Our results indicated that administration of PTX early after the onset of sepsis prevented the decrease in AM responsiveness at both the macro- and microcirculatory levels in late sepsis [27]. However, the elevated plasma levels of AM were unaffected by PTX treatment [27], indicating that the beneficial effects on vascular responsiveness was not mediated by altering AM. Furthermore, increased levels of plasma TNF-␣, IL-1␤, and IL-6 observed during late sepsis were attenuated by PTX administration, suggesting that maintenance of AM responsiveness by this agent appears to be due to downregulation of these inflammatory cytokines [27]. It is therefore suggested that treatment with PTX is an effective approach for maintaining vascular reactivity in the late stage of sepsis. Although it has been shown that AM and its pharmaceutical ligands appear to be useful in the treatment of cardiovascular diseases [12,33,34], we propose that pharmacologic modulation of the vascular responsiveness to AM such as the use of PTX may also be important in the maintenance of hemodynamic stability during the progression of polymicrobial sepsis. Future areas of investigation should determine the mechanisms underlying the reduction in vascular reactivity during sepsis as well as the alterations involving AM receptors and binding proteins under those conditions. Specifically, studies are proposed to determine: 1) whether AM gene delivery prolongs the hyperdynamic response during sepsis; 2) whether one of the mechanisms responsible for the protective effects by AM is due to an increase in vascular endotheliumderived NO release; 3) whether the protective effects of AM are contributed by the maintenance of vascular responsiveness to AM stimulation; 4) whether AM downregulates proinflammatory cytokine (TNF-␣, IL-1␤, IL-6) production following the onset of sepsis. Future studies and developments in sepsis research will lead to improved management of septic patients and decreased morbidity and mortality. Acknowledgments The author wishes to express his sincere thanks to David Ornan for his excellent assistance in preparing the manuscript. This work was supported by the National Institutes of Health Grant RO1 GM 57468 and KO2 AI 01461. References [1] Asada Y, Hara S, Marutsuka K, et al. Novel distribution of adrenomedullin-immunoreactive cells in human tissues. Histochem Cell Biol 1999;112:185–91.

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