Considerable efforts have been expended to find novel therapeutic strategies to combat sepsis. Unfortunately, most of these strategies failed to improve patient survival when studied in large, multicenter clinical trials 19,20
. Many of the pathways which were targeted, e.g., IL-1 and TNF-α, are part of an extensive redundant network that cannot be interrupted with an agent that blocks a single pathway. Only activated protein C, with its its extensive effects on the coagulation cascade and on downstream inflammatory mediators, has shown any promise in the treatment of sepsis21
. Our goal was to find a treatment for sepsis utilizing our experience with lipoproteins and their unique role in inflammation.
Apolipoprotein E plays a role in increasing the immune response to polymicrobial sepsis, particularly through increased NKT cell number, proliferation, and downstream responses, which contributes to mortality4
. ApoE thus provides a potentially valuable upstream target for therapeutic intervention that can exert a more global effect on inflammation. Heparin, an established anticoagulant, fulfills the role of apoE antagonist with its affinity for the LDLR-binding portion of apoE. This study provides evidence that heparin can reduce hepatic NKT proliferation and peripheral cytokine production that occurs in response to sepsis, and thereby contributes to decreased mortality. Our in vitro
data points to an LDLR-dependent mechanism by which heparin prevents apoE internalization. In addition, heparin variants serve as controls in our efforts to prove that heparin decreases septic mortality independent of its anticoagulant activity.
Unfractionated heparin clearly increased survival of mice subjected to cecal ligation and puncture compared to saline. 20 U/kg/hr, equivalent to the dose given for the pulmonary embolism protocol, conferred the most survival benefit. To date, animal studies looking at heparin effects during sepsis have been mixed. These studies utilize various methods to induce sepsis, including intravascular e.coli infusion, intraperitoneal meningococcal or fecal injection, and biliary fecal injection14,22–24
. Our chosen sepsis model, cecal ligation and puncture, has been proven to be both highly reproducible and clinically relevant25
. Clinical studies looking at heparin effects in septic patients have produced mixed results. A retrospective Canadian study demonstrated a reduction in 28 day mortality from 69% to 56% in patients given at least one day of heparin therapy for various indications compared to those not given heparin.26
In contrast the first randomized single center trial looking at heparin administration following signs of sepsis (the HETRASE Study) showed no significant change in 28 day mortality (16% vs 14%) when compared to the control group27
. The different outcomes of these two clinical studies could be attributed to the different patient populations in terms of disease severity in addition to the dosage of heparin given. But the inability to demonstrate consistent survival benefit still put into question the assumption that heparin primarily acts in sepsis to counteract the DIC response, specifically to deplete the potent pro-inflammatory generators thrombin and fibrin.
Interestingly, our study showed that anticoagulation was not necessary for the observed survival improvement. When heparin was administered at high doses, 80 U/kg/hr, the survival benefit was minimal even though this group was effectively anticoagulated 24 hours after surgery. In addition, octasaccharide, a heparin variant which has only a fraction of the anticoagulation potency of heparin but similar apoE-binding ability,7,12
demonstrated a survival benefit in sepsis without obtaining significant anticoagulation. These results are in line with a recent prospective randomized clinical study out of China demonstrating a 28-day survival benefit in septic patients given heparin when compared to the control group (15.4% vs. 32.4%), but no difference in PTT.28
Similarly, the subgroup analysis of the antithrombin III Kybersept trial showed that in moderate risk septic patients, high dose ATIII therapy decreased the 28-day mortality versus controls (44.4% vs. 35.7%) without a significant change in PTT, PT, or fibrinogen.29,30
These findings support previous clinical and animal study findings that allude to the fact that the anticoagulation, anti-DIC effects of heparin are insufficient for septic protection27,31,32
. The subgroup analysis of the antithrombin III Kybersept trial demonstrates that moderate risk patients We believe that a significant component of the survival benefit seen with heparin can be attributed to its apoE-binding properties. This theory was further corroborated by our finding that lepirudin, which decreases fibrin and thrombin production but does not bind apoE, effectively anticoagulated septic mice at high doses but failed to demonstrate any improvement in survival and in fact increased overall mortality.33
Perhaps the lack of apoE antagonism is what makes lepirudin an ineffective anti-sepsis agent.
Our hypothesis is based on the aforementioned mechanism whereby apoE facilitates lipid antigen presentation in an LDLR-mediated proinflammatory cascade (), and heparin binding of apoE can interfere with this process. In order to define the specific interaction between heparin and apoE, we attempted to monitor what happens to endogenous apoE with the addition of heparin. We found that in the presence of a fixed concentration of apoE, heparin decreases LDLR-mediated uptake of apoE in fibroblasts. This interaction supports the mechanism proposed by van den Elzen et al. and our in vivo
findings that heparin can diminish apoE-mediated lipid antigen presentation and ultimately downregulate the LDLR pathway that leads to inflammation and death.5
The concept of heparin interacting with apoE and the LDLR is not a new one. In the liver, apoE mediates lipoprotein remnant binding to the heparan sulfate proteoglycan (HSPG) – LDLR-related protein pathway, facilitating liproprotein uptake.34
ApoE contains a C-terminal domain which contains the major lipid-binding (and presumably lipid antigen–binding) site, and a N-terminal domain which contains the LDLR-binding region. This N-terminal domain also contains a high-affinity heparin-binding site overlapping with the receptor region.7,9
Conceivably, heparin could bind the N-terminal domain of apoE and interfere with its ability to bind the LDLR either by directly blocking the site or by inducing a conformational change that prevents it from binding the receptor. The C-terminal domain of apoE also contains a lower affinity heparin-binding site.9
Heparin could similarly decrease the lipid antigen-binding potential of the apoE C-terminal domain, and thereby reduce lipid antigen presentation and inflammation. Regardless of the specific pathway, decreasing apoE--LDLR or apoE--lipid antigen could both decrease downstream CD1d-restricted NKT cell activity and cytokine release.
NKT cells, which are predominantly expressed in the liver, appear to act as a bridge between the innate and acquired immune systems. Hepatic NKT cell frequency decreased significantly in septic mice treated with heparin when compared to septic mice given saline. ApoE has been shown to increase CD1d-restricted NKT cell activity in sepsis4
. We speculated that the addition of the presumed apoE-antagonist, heparin, could decrease NKT proliferation. Not only did heparin decrease NKT cell frequency, but it also decreased ALT levels which we interpreted as an index of apoE-induced NKT cell hepatoxicity. Upon stimulation, NKT cells can promptly secrete large amounts of Th1 and Th2 cytokines. Previous research suggests that both pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ, as well as anti-inflammatory cytokines such as IL-4 and IL-10, have relevant roles in sepsis35
. ApoE activation of NKT cells appears to produce a mixed cytokine response, with Th1 cytokines predominating (IL-10, TNF-α, IL-1β, and IFN-γ)4
. All these cytokines were reduced in septic mice treated with heparin when compared to saline treated mice. IL-4, which is the main Th2 cytokine produced by NKT cells, was also reduced with heparin administration. Both the NKT cell and cytokine decreases appear to be dose-specific, with only high dose heparin resulting in a significant reduction. Interestingly, although both low and high dose heparin decreased hepatic NKT cell frequency and cytokine production, only high dose resulted in a significant reduction when compared to untreated septic mice. This is in contrast to our survival data, in which the low dose heparin demonstrated optimal survival benefit compared to high dose heparin. One possible explanation is that heparin is acting through an additional pathway that does not involve NKT cells. Another possibility is that unlike high dose heparin, low dose heparin achieves the optimal balance of minimizing the detrimental hyperinflammatory response without being overly immunosuppressive.
We acknowledge that heparin is a diverse molecule that has the potential to exert an array of anti-inflammatory effects. From inhibition of leukocyte adhesion36
, to selectin and PECAM binding37–39
, heparin has been revealed to be much more than just an anticoagulant. Although how these functions play out in in vivo
has not been completely elucidated. These alternate functions could very well exist in parallel to the mechanism that we feel makes a significant contribution to septic mortality. Our focus is to highlight the significant role that apoE-mediated immunomodulation plays in septic death and how heparin acts to counteract these detrimental effects. In order to define heparin’s specific role in the apoE-mediated inflammatory response, we will have to repeat our in vivo
experiments in apoE, LDLR, and NKT (Jα-18) knockout animals. These future experiments will allow us to isolate the integral nature of each component of the pathway. Why heparin to date has not consistently shown survival benefit in sepsis is still to be determined. It is possible that the timing of the treatment is critical to significantly blunting the systemic inflammatory response before the cascade propagates out of control. In our animal study, we have the luxury of initiating treatment at the time of the insult. Further studies are necessary to address the therapeutic window for heparin in sepsis.
In conclusion, our study provides evidence that heparin can decrease inflammation and mortality in a murine model of sepsis independent of its traditional anti-thrombotic effects. Research has highlighted a growing role for agents of lipid metabolism, specifically apoE, as playing a significant role in foreign lipid antigen processing and in the immune response. Heparin binds with high affinity to apoE, and this unique interaction leads to downregulation of the LDLR-mediated hyperinflammatory cascade. Ultimately, the goal will be to find an agent that can minimize the pro-inflammatory effects of endogenous apoE without the potential side effect profile of heparin. Potential candidates include heparin variants such as octasaccharide, apoE-mimetic peptides, which can occupy LDLR sites without facilitating lipid antigen presentation, apoE-antibodies, and soluble apoE-receptors. Our research highlights the importance of apoE in regulating infection and immunity, and the importance of cultivating this role for future sepsis therapy.