SPLANCHNIC VASCULAR CONTROL DURING SEPSIS AND ENDOTOXEMIA

Albert D. Sam II¹, Avadhesh C. Sharma², William R. Law^{1, 2} and James L. Ferguson²

Departments of Surgery¹ and Physiology & Biophysics^2, The University of Illinois College of Medicine at Chicago 835 South Wolcott Avenue, Chicago, IL 60612-7342

Received 7/2/97 Accepted 9/22/97 4. REGULATION OF VASCULAR RESPONSES DURING THE PROGRESSION OF SEPSIS

Endothelium - formerly thought to be an inert structure - is now recognized as an endocrine/paracrine structure which secretes several vasoactive mediators. The state of the vascular endothelium during sepsis is paramount in assessing the vascular mediation in maintaining vascular tone. The control of vascular tone is dependent on neural factors, humoral factors, mechanical forces and the physiological milieu of the endothelium (Figure 1). These factors acting either directly on vascular endothelium, or indirectly through other substances affect the diameter of the resistance vessel. To date a large number of these factors have been identified (34); however, the mechanism of vascular control during endotoxemia and sepsis is not well understood. Several studies addressing this issue have centered on the endothelium's ability to produce endothelium-derived vasoactive factors as a primary determinant of vascular fitness (35,36). Our laboratory has emphasized the influence of these neural, humoral (adrenergic), endothelial factors (nitric oxide and endothelins) and cellular metabolic products (adenosine) in the maintenance of blood perfusion during endotoxemia and more recently during sepsis. In this review, we will explore the advancements made in this area and clarify the current information regarding the underlying mechanisms responsible for sepsis-induced vascular derangements in the splanchnic circulation.

Figure 1. Mechanical forces and pathophysiological conditions (including sepsis and endotoxemia) cause activation of endothelial cells in an autocrine or endocrine manner releasing a myriad of vasoactive mediators: cytokines, endothelins (ET), nitric oxide (NO), etc. Other tissue metabolites (adenosine) act directly on smooth muscle to induce vasodilation (not shown in figure). The biological algebraic sum of these vasoactive mediators [vasoconstriction (ETs) and vasodilation (NO, adenosine)] determine vascular tone. 4.1 Cytokines Cytokines are soluble polypeptides elicited in a number of pathophysiological states including inflammation, septic shock and trauma. Although traditionally known to modulate the immune system, their effects are not limited exclusively to the components of the immune system. These polypeptides are produced by a variety of cells, including monocytes, macrophages, lymphocytes, epithelial, endothelial and parenchymal cells of the gastrointestinal tract. In normal physiology, cytokines function at low levels and help to maintain homeostasis. However, after any inflammatory insult, these mediators are produced at the site of injury by a large number of cells and affect the biological system.

Even though our understanding of the host response to endotoxemia/sepsis has progressed steadily, the precise role of the individual cytokines-particularly as vascular mediators-is extremely limited. It is well known that the majority of the physiologic, metabolic, and immunologic responses to immune challenges are not due to endotoxin but rather cytokines (Figure 2). These proteins operate in concert with hormones and humoral mediators to elicit and orchestrate the cellular response to sepsis/endotoxemia. Cytokines bind to specific plasma membrane receptors resulting in gene expression which both augments and attenuates the immune response. The effect of cytokines on peripheral vascular tone is well known as hypotension often results after cytokine induction. However, direct splanchnic vascular mediation due to cytokine release has not been firmly established to date.

Figure 2. The cytokine signaling-response system: tumor necrosis factor (TNF), interleukins (IL), and interferons (IFN) are produced after stimulation by endotoxemia, bacteria or peritoneal sepsis. The Cytokine producing cells are: lymphocytes, macrophages, fibroblasts, etc. The effector cells are endothelial cells, hepatocytes

Though cytokines are not directly involved in controlling vascular responses (37), they are now known to induce the production of vasoactive mediators. In the following sections, we will briefly review some of the cytokines in relation to their role in splanchnic vascular control.

4.1.1 Tumor necrosis factor-alpha mediated mechanisms

Tumor necrosis factor (TNF)-alpha, produced primarily by activated macrophages and helper T-cells (38-40) is the most proximal cytokine mediator. It is detected in the serum within 20 minutes after an immune challenge (41). Its concentration peaks between 90 minutes and 2 h after endotoxin injection (42). TNF-alpha has a half-life of 14-18 minutes (43) and is degraded in the liver, skin, GI tract, and kidneys (41). Some of its actions are to increase the production of other cytokines and growth factors, release of neutrophils from bone marrow (44-47), mobilization of fat stores and amino acids in muscle (48-51) resulting in catabolic wasting, increasing the expression of intracellular adhesion molecules (ICAM)(52), and increased production of inducible nitric oxide (53-57). Studies have shown that TNF- alpha in septic patients usually has its highest concentration upon hospital admission and may remain elevated in those patients that eventually succumb to multi-system organ failure (58). For this reason, it is advantageous to assess its role in splanchnic vascular mediation.

Recombinant TNF-alpha administration in non-septic animals has been shown to cause vascular endothelial cell dysfunction without reducing cardiac output and tissue perfusion (59,60). The administration of a TNF- alpha inhibitor prior to the onset of sepsis in the rat model has been suggested to maintain aortic ring vascular structure and function (61). Whether these findings may be extrapolated to changes prevalent in the microvasculature remains controversial.

TNF-alpha may adversely affect splanchnic perfusion by its ability to induce the expression of ICAMs on the endothelial cell surface. These molecules may impede bloodflow due to the changes that may occur in viscosity as a result of their production. Vascular endothelial cells are located between blood and tissues and are vital in the action of cytokines (52,62). ICAMs serve as "glue" for intercellular attachment. A soluble form of ICAMs (sICAM) is released into the bloodstream b, monocytes, neutrophils, and endothelial cells during sepsis (63). Endotoxin and inflammatory cytokines have been shown to quantitatively increase the number of adhesion molecules on vascular endothelium and in the circulation. Significant correlation has been shown between sICAM-1 levels, TNF-alpha and endotoxin (52,62). This evidence suggests that the rheological changes in the circulation possibly induced by TNF-alpha due to ICAM production may result in an overall impairment in splanchnic perfusion by increasing the viscosity of the bloodstream.

4.1.2 Interleukin - mediated mechanisms

Shock and its sequela can be demonstrated when IL-1 is administered alone or in combination with other pro-inflammatory cytokines (64). IL-1 binds to specific cellular receptors designated as type I and type II. The Type I receptor is a transmembrane glycoprotein with a molecular ratio (Mr) of 80,000. It is a member of the Ig- protein family with three Ig-SF C2 set domains (65,66). This receptor is important in signal transduction after interleukin binding (66). The type II receptor is a glycoprotein with an Mr of 60,000, with three Ig-SF C-2 set domains in its extracellular region, a 29 amino acid cytoplasmic domain and a single transmembrane peptide segment (65,66). This receptor does not lead to signal transduction mechanisms and may serve as a decoy to reduce the amount of IL-1 available to bind the type I receptor (66,67). Since arterial and arteriolar structural integrity is vital to vascular regulation, and since endothelial derangements occurs in states of severe shock, the effects of interleukin on the vasculature is important in determining its role in splanchnic vascular control.

A novel study by Sutton et al Examined the structural integrity of endothelial (aortic) cells after endotoxemia in normal animals and in those genetically devoid of the type I IL-1 receptor (68). Ultrastructural comparisons revealed that the knockout mice treated with endotoxin showed total maintenance of endothelial structural integrity as compared to wild type animals receiving endotoxin (68). These findings support previous work performed by Norman et al. who showed that IL-1 antagonism during a lethal sepsis model in rodents exerts a protective effect by maintenance of aortic endothelial architecture and maintenance of vascular tone (69).

One potential mechanism of endothelial damage due to IL-1 is speculated to be its ability to induce the production of IL-6. IL-6 is elevated in septic shock and has been shown to correlate with clinical outcomes in some animal studies (64). Sutton's study also monitored IL-6 levels and demonstrated a correlation between endothelial damage and IL-6 concentrations. In wild-type control animals treated with endotoxin, an 87-fold increase in the serum level of IL-6 and endothelial damage was observed. The knockout animals, which exhibited a 5-fold increase in IL-6, showed no endothelial damage (64). A subsequent increase of the endotoxin dose by 5-fold in wild-type control animals resulted in a 94-fold increase in IL-6 concentration yet without producing endothelial damage. This shows that, in this model, there appears to be no correlation between IL-6 levels and the compromise of the endothelial integrity.

4.2 Complement - mediated mechanisms

TNF-alpha alone and in synergy with endotoxin has been shown to also activate the complement pathways. Certain bacterial cell wall lipopolysaccharides directly activate the alternative pathway (38). The fifth component of C5 becomes cleaved and activated during the complement cascade giving rise to C5a. This is rapidly converted by serum carboxypeptidase N to C5a des Arginine - thought to be the mediator (or co-mediator) of the resultant systemic vascular effects. C5a, an alternative complement component and significant peripheral vascular mediator during inflammatory states, is well-known to exert a vasodilating effect on the vasculature (70). In rats, endotoxin bolus results in an increase in C5a concentration which reach baseline values after 30 minutes (70). The resultant arterial hypotension was attenuated with the administration of anti-C5a antibodies - a non-polymorphonuclear-leukocyte effector.This implicates a causal relationship. Since indomethacin inhibition abolished the hypotensive response it was hypothesized that C5a - induced hypotension occurs via a prostanoid-mediated mechanism (70).

The effect of C5a on splanchnic vasomotor regulation during sepsis or endotoxemia has, to our knowledge, not been completely characterized. Lundberg et al assessed regional hemodynamics in nonseptic rats after C5a challenge and observed a reversible systemic hypotensive response associated with the release of vasodilating and vasoconstricting products of the cyclooxygenase pathway (71). In addition, they reported a 25% decrease in both cardiac output and hepatic blood flow after C5a administration (71). They attributed their findings of arterial hypotension to dual mechanisms involving a thromboxane A2 - induced vasoconstriction in the pulmonary bed resulting in decreased cardiac output and a prostaglandin I2 - mediated peripheral vasodilation. Although extrapolation of these findings to septic models must be viewed with caution, this study suggests that some aspects of splanchnic vasoregulation is mediated through the alternative complement pathway.

4.3 Adenosine - mediated mechanisms

Adenosine is recognized as a potent vasodilator. Adenosine is produced in numerous tissues after the degradation of AMP by 5' nucleotidases (72). Ubiquitous in physiological systems, it plays an active role as a regional regulator of tissue perfusion via a receptor-mediated process which produces vasodilation in hypoxic tissues and redistributes blood within hypoxic vascular regions (73,74). In an intravenous E.coli septic model in rats, quantitative changes in the adenine nucleotide pools were shown in the liver and small intestine (75). Even though direct measurements of adenosine and/or its catabolites in septic tissues was not done, this has been performed in rats subjected to hemorrhagic shock and significant elevations of adenosine and catabolite products were observed (76).

The effects of adenosine on intestinal micro-circulation has been delineated by Granger and Norris (77). Using a canine model, they reported that theophylline, an adenosine receptor antagonist, significantly reduced mesenteric flow responses in chow fed animals and no effects in unfed animals. This suggests an augmented role of adenosine during an increase in metabolic activity.

Evidence from our lab has shown that adenosine is actively involved in the redistribution of blood flow toward the splanchnic organs and skeletal muscle during sepsis and during an increased metabolic activity (78). We have shown that blockade of adenosine receptors in rats during sepsis, utilizing the non-selective adenosine receptor antagonist, 8-phenyl-theophylline (8-PTH), resulted in an increase in hepatic-portal and skeletal muscle vascular resistance (Figure 3) as compared to non-septic controls (78). In addition, an organ-specific effect was seen in septic and nonseptic animals treated with 8-PTH. When compared to its vehicle, 8-PTH caused a significant decrease in flow to small intestine, cecum, colon, and pancreas in septic rats which were not seen in non-septic controls.

Figure 3. Hepatic portal and skeletal muscle resistance in septic and non-septic rats after treatment with 8-PTH or vehicle.

These results imply that there is an increased production of adenosine during the septic state which can be attributed to two pathways: (1) a reduction in the tissue oxygen tension due to either increased oxygen supply or increased consumption; or (2) increased adenyl cyclase activity resulting in increased adenosine production from cAMP degradation providing substrate for adenosine formation. Alterations in oxygen supply-demand dynamics is critical in the pathogenesis of sepsis. Adenosine, on-the-other- hand, an important energy metabolite, is an important modulator of perfusion to the hepato-splanchnic circulation during sepsis.

4.4 Nitric oxide - mediated mechanisms

Nitric Oxide (NO), a potent vasodilator, is produced by vascular endothelial cells from its precursor L-arginine which is subsequently converted to citrulline by nitric oxide synthase ENOS (Figure 4). NO, by virtue of affecting vascular smooth muscle guanylate cyclase mediates the vasodilating effect. NOS exists in three isoforms characterized by their pattern of activity (i.e. constitutive vs. inducible) and their requirement for calcium. Types I and III are located in the nervous system and endothelium respectively and are classified as constitutive, calcium dependent isoforms. Type II is located primarily in macrophages and is classified as an inducible, calcium independent isoform. Type II NOS is stimulated by septic mediators such as endotoxin and cytokine (79) and requires 4-6 hours for expression after an immune challenge (80). Due to its association with calmodulin, quantitatively more NO is produced from this isoform than the other two (nanomoles vs. picomoles) (81).

Figure 4. Synthesis and actions of nitric oxide (NO): NO synthase exists in three isoforms: NOS I & III are constitutive producers of NO. NOS II is the inducible isoform. (CO: cardiac output).

Numerous studies have attempted to inhibit nitric oxide production by NOS during sepsis. Most have reversed sepsis-induced hypotension yet uniformly resulted in poor mortality outcomes (82-88). Rubin et. al. harvested aortic endothelial cells from guinea pigs 16 h after induction of endotoxemia. The production of NO, in a concentration-dependent manner, decreased after exposure to endothelium receptor-dependent agonists

and not endothelium receptor-independent agonists (89). Thus, the use of NOS inhibitors during this time period in sepsis may further compromise NO production leading to vascular tone derangements and compromised tissue perfusion (89-91).

Inhibition of cNOS by L-NMMA (nitro-guanido-monomethyl-L-arginine) reduces splanchnic perfusion and exacerbates intestinal vascular injury induced by endotoxin challenge (92,93). More recently, the focus of attention has shifted to the inhibition of iNOS since this isoform is selectively increased during sepsis and, quantitatively, has a greater significance (94). Several compounds have been reported to inhibit iNOS, however, their selectivity remains controversial. Data from this lab assessed the ability of aminoguanidine in attenuating NO production after chronic sepsis in rats. After 30 minutes of infusion, aminoguanidine led to a significant decrease (65 micromolar vs. 90 micromolar) in the serum nitrates. The serum level of nitrates returned to previous septic levels once infusion was terminated (95). Further studies in our lab have elucidated an important role of the inducible form of NOS in regulation of perfusion during sepsis. Administration of aminoguanidine resulted in significant decreases in perfusion to ileum in septic rats compared to controls (unpublished data).

It also has been known that septic individuals and endotoxemic or septic animals demonstrate impaired responsiveness to sympathetic nerve stimulation and to exogenously administered adrenergic agonists. Recent evidence has implicated NO as a main factor in the generation of this vascular unresponsiveness (96). Since it has been shown that vascular responses to calcium were affected during sepsis, it is thought that a defect in contractile machinery is responsible for some of the unresponsiveness and to the hypotension during sepsis. Ex vivo aortic preparations have displayed endothelium-independent responsiveness to norepinephrine which is restored by the addition of NOS inhibitors (96) implicating NO as an important contributor to septic-induced vascular derangements. Additional evidence for the role of NO stems from estimation of cGMP demonstrating a significant elevation in vessels removed from endotoxemic rats as compared to controls (97).

In vivo studies in endotoxemic rats assessing pressor responses to norepinephrine resulted in reductions of responsiveness as assessed by changes in the arterial blood pressure. These alterations were restored to normal levels following the administration of the NOS inhibitor L-NMMA (96). This evidence from In vivo and ex vivo studies suggest that NO plays an important role in changes in the vascular tone during sepsis which may be inferred to also occur at the splanchnic vasculature.

Hemoglobin is a well-recognized scavenger of nitric oxide. Its actions are in part through a NO-mediated pathway independent of NO synthesis. A recently developed and modified hemoglobin based blood substitute, diaspirin cross-linked hemoglobin solution (DCLHb) has been shown to interact closely with NO (98) and Endothelins (ET) (99,100), potent vasoconstrictors. DCLHb, a stroma free, non-antigenic, oxygen-carrying molecule, has been shown to improve regional blood flow in the treatment of hemorrhagic shock (101-103). DCLHb has pressor qualities independent of alpha-adrenergic pathways (104-107).

In our laboratories, studies were performed with the hypothesis that DCLHb infusion would improve organ perfusion in cecal-slurry induced septic rats. Rats received either 100 or 250 mg/kg DCLHb or albumin at 1, 2, or 4 h after induction of sepsis. Moribund rats were identified and received 100 mg/kg DCLHb or iso-oncotic albumin infusions at identical time points. Radiolabled isotopes were infused pre-sepsis induction, pre-DCLHb infusion and post- DCLHb infusion to assess changes in tissue blood flow. In rats that received DCLHb, all regions of the GI tract demonstrated significant increases in perfusion 24 h after sepsis induction (32). Perfusion to the kidneys, pancreas, and liver were not significantly altered. This particular study also showed an increase in systemic vascular resistance after treatment but not at the expense of specific organ system perfusion - the ultimate goal of resuscitation therapy.

Anecdotally, DCLHb treated rats displayed reduced peritoneal extravasation at necropsy compared to albumin treated rats. Cecal contents also were observed to appear unusually dry. Together, this may indicate that modified hemoglobin (DCLHb) may provide protection to intestinal vascular beds and prevent the loss of vascular fluid to the third space compartments in the form of ascites. However, further studies are still needed to confirm that this protective effect of hemoglobin preparation is due to its interactive role with NO and or other (i.e. endothelin) mechanisms.

4.5 Endothelin - mediated mechanisms

Endothelins (ET), potent vasoconstrictors also produced by vascular endothelium, have recently gained acceptance as important mediators of vascular tone (108), particularly in sepsis. They are 21-amino acid peptides consisting of ET-1, ET-2, ET-3, and vasoactive intestinal contractor (VIC). ET-1 gene expression is up regulated by endotoxin or LPS in human macrophages (109). Cytokines have also been shown to affect ET gene expression (109).

Produced from the cleavage of 203-213 amino-acid proteins called preproendothelins into precursors called big endothelins (big ET-1, big ET-2, big ET-3), their final conversion is dependent on metalo-endoproteases called endothelial converting enzymes (Figure 5) generating ET-1, ET-2, or ET-3. Three isoenzymes (ECE-1a, ECE-1b, and ECE-2) have been isolated to date. ET's binding directly to their specific receptor results in alterations in vascular tone. These G-protein-coupled receptors - ETa, ETb, and ETc - elicit responses that are dependent upon the concentration ratios of ET's: ETa receptors are activated when ET-1 is equipotent to ET-2 but more potent then ET-2 (1=2>3); ETb receptors are activated when ET-1=ET-2=ET-3 (1=2=3); ETc receptors are activated when ET-3>ET-1 or ET-2 (3>1,2) (110-112).

Figure 5. Role of endothelin pathways in sepsis and endotoxemia. aa=amino acid, ECE=endothelin converting enzyme, proET (big endothelin)

As described above, besides ET's own independent effects on vascular tone, ET production is keenly interrelated with other mediators during sepsis. An exogenous challenge of ET induces the release of prostanoids (thromboxane A2 and prostacyclin) and nitric oxide (113) among others (114). Additionally, cytokines (TNF-alpha, IL-1alpha, IL-1beta, IL-2 or IL-6) also cause increased production of ET and release from vascular endothelial cells. Even though stimulants for ET production are most likely multi-factorial, TNF-alpha appears to be required for its release. Administration of anti-TNF antibodies prior to TNF-alpha infusion in rats (115) or LPS treatment in pigs (116) significantly reduced circulating ET-1 levels.

The role of ET in sepsis has been implicated by several observations: Plasma ET concentrations are elevated in septic patients (117); plasma ET levels double during sepsis in rats (118); ET-1 anti-serum improves hemodynamics and alleviates shock injury in septic animals (119). These studies demonstrate the potential of ET-mediated mechanisms in the pathophysiology of sepsis. The profile of ET production at various time points during sepsis and whether varied release of ET alters the progression of sepsis is unknown.

In a recent study, we hypothesized that induction of intraperitoneal sepsis would not only alter myocardial performance but also the circulating and myocardial concentrations of ET and NO during sepsis. This study was undertaken to determine 1) myocardial performance at 24 and 48 hours following sepsis/sham sepsis, and 2) the profile for ET and NO in the plasma and left ventricular tissue during sepsis. The results indicated that progression of sepsis in our rat model may occur at least in two phases. Phase 1 (early phase, 0-12 h after induction of sepsis), when both plasma ET and NO showed an increase in response to induction of sepsis and phase 2 (late phase, 12-48 h after induction of sepsis), when plasma ET levels returns to basal levels while NO remained elevated. Thus, these two potent vasoactive agents have a divergent time course that is likely to be related to the different mechanisms of control of the vascular tone during sepsis (120). The implication of this observation on the regulation of splanchnic perfusion has not been assessed directly, however, it is likely that ET - mediated mechanisms play a significant role in splanchnic vasomediation during sepsis.