Hemorrhagic shock (HS) can be a lethal disease, with more than 60,000 deaths per year in the United States and an estimated 1.9 million deaths per year worldwide. Acute massive blood loss induces excessive inflammatory cytokine production and inflammatory cell activation with subsequent systemic inflammatory response syndrome. The abnormal inflammatory response leads to organ damage and multi-organ dysfunction syndrome. At the cellular level, HS results in cell death due to hypoxia-reperfusion injury, and subsequent inflammatory insult (1, 2).
Current management of HS mainly focuses on hemorrhage control and resuscitation but does not include pharmacologic agents targeted at improving cell survival. Our team has focused on identifying pharmacologic therapies that can protect the cells during shock and improve survival.
Peptidylarginine deiminase (PAD) is a calcium-dependent enzyme which can convert positive arginine residues on cellular proteins into neutral citrulline residues. This post-translational modification, called citrullination or deimination, can influence various protein functions. The PAD family consists of five isoforms including PAD 1, 2, 3, 4, and 6 with tissue-specific distribution. PAD plays an important role in inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and neurodegenerative diseases (3–5). Hemorrhage can also lead to widespread inflammation, especially after resuscitation and associated ischemia-reperfusion (I/R) injury. A series of studies have shown that PAD4 activation exacerbates kidney I/R injury (6), Pad4−/− mice are protected against kidney and liver injury after renal ischemia-reperfusion (7), and Pad4−/− improves survival and a lethal two-hit model of HS and sepsis (8).
We have previously tested a combination PAD2/PAD4 inhibitor (YW356) in HS, and discovered that its administration attenuates inflammation and improves survival in a rat model of hemorrhagic shock (9). That was the first study demonstrating that PAD play an important role in HS, but it was not designed to identify the relative roles of the two PAD isoforms. Hence, in the present study, we utilized selective Pad−/− knockout mice to understand if Pad2−/− improves survival in HS, and to identify potential underlying mechanisms.
MATERIALS AND METHODS
This study conformed to the Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee. Pad2−/− mice were kindly provided by Dr Scott Coonrod (Cornell University). Pad2−/− and wild-type (WT) mice were FVB/NJ strain. WT mice were purchased from Jackson Laboratory (Bar Harbor, Maine), were used for their respective control. We only used female animals in this experiment to eliminate gender dimorphism as a confounding variable.
Hemorrhagic shock models
Hemorrhagic shock was induced in a mouse model, as described previously (10). All experiments were performed at the same time by the same operator (JZ). Mice were anesthetized with 5% isoflurane, and maintained via a nose cone by delivering 1.6% to 2.5% isoflurane using a veterinary multichannel anesthesia delivery system (Kent Scientific Corporation, Torrington, Conn). Bupivacaine (1%) was injected subcutaneously at the operative site for analgesia. The left femoral artery was dissected and cannulated with polyethylene catheters 10 (Clay Adams, Sparks, Md) which were primed with heparinized saline (10 USP U/mL).
In the lethal experiment, mice (n = 5 per group) were subjected to hemorrhage of 55% total blood volume over 10 min. The volume of hemorrhage was calculated using the following formula: Estimated blood loss volume (mL) = body weight (g) × 0.07 (mL/g) × 0.55 (10). After hemorrhage, cannulas were removed, femoral arteries ligated, and skin incision closed. Animals were survived for 7 days prior to the predetermined endpoint and euthanasia. Animals were left unresuscitated for the entirety of the experiment.
The markedly different survival rates in the lethal hemorrhage model precluded time-matched tissue sample procurement. This prompted us to perform mechanistic studies in a non-lethal model of HS. WT mice and Pad2−/− mice (n = 3) were subjected to 30% total blood volume hemorrhage of over 5 min. Sham animals were anesthetized and underwent femoral cannulation without hemorrhage. Twelve hours after hemorrhage, animals were euthanized for serum and tissue harvest. Tissue samples for histological analysis were fixed in 4% formalin. Serum and tissue samples for cytokine analysis and Western blot analysis were snap-frozen in liquid nitrogen immediately and then stored in −80°C.
Myocardial infarction model
To investigate why Pad2−/− mice had improved survival in the lethal HS model and the specific effect of Pad2−/− on cardiac function, WT and Pad2−/− mice were subjected to myocardial infarction (MI) induced by permanent ligation of the left anterior descending artery (LAD) of the heart, as described previously (11, 12). Echocardiography and triphenyltetrazolium chloride (TTC) staining were performed 24 h after MI. After TTC staining, the infarct size was calculated as the percentage of ischemic area (IS, white region) versus area at risk (AAR, non-blue region) (IS/AAR). AAR was calculated as a percentage of the cross-sectional area of the left ventricle (LV) (AAR/LV). Echocardiography was performed using a Vevo 770 system 24 h after MI. Ejection fraction (EF) and fractional shortening (FS) were measured by a blinded investigator.
Acute lung injury scoring
Lung tissues from the non-lethal HS model initially fixed in formalin were embedded in paraffin and sectioned. After deparaffinization and dehydration, the sections were stained with hematoxylin and eosin (H&E). The H&E stained slides were examined by an experienced histopathologist (XD) who was blinded to the group allocation of the samples. Slides were scored for the severity of acute lung injury (ALI). ALI scores consisted of six parameters: septal mononuclear cell/lymphocyte, septal hemorrhage and congestion, neutrophils, alveolar macrophages, alveolar hemorrhage, alveolar edema. The total scores were calculated by adding the scores of each parameter (13).
Inflammatory cytokines analysis
Serum levels of cytokine-induced neutrophil chemoattractant 1/keratinocyte-derived chemokine (CINC-1/KC) were measured by enzyme-linked immunosorbent assay according to the manufacturer's protocol (R&D Systems, Minneapolis, Minn).
Western blot analysis
For β-catenin analysis, proteins were extracted from the heart tissues using radioimmunoprecipitation assay buffer with protease and phosphatase inhibitor (Thermo Scientific, Rockford, Ill). Equal amounts of proteins were separated by 12% SDS-PAGE and followed by transfer onto a nitrocellulose membrane. The membrane was blocked with 5% milk, incubated at 4°C overnight with mouse anti-β-catenin polyclonal antibody (Abcam, Cambridge, Mass), diluted 1:1,000 in 5% bovine serum albumin, washed and incubated with rabbit antimouse IgG antibody or goat antirabbit IgG antibody (Abcam, Cambridge, Mass) for 2 h at room temperature. Chemiluminescence detection was performed by using chemiluminescent substrate (Thermo Scientific, Rockford, Ill) and imaged by ImageDoc MP Imaging System (Bio-Rad, Hercules, Calif). Quantitative analysis of detected bands was conducted by densitometer scanning using ImageJ (NIH, Bethesda, Md).
Kaplan–Meier method curves, with log-rank testing, were used to calculate survival differences between the groups. One-way analysis of variance was used for comparing infarct size, EF, FS, ALI scores, and serum CINC-1/KC among the groups. Tukey's post hoc testing was used for comparing intergroup differences. P value < 0.05 was considered significant. Prism 6 (GraphPad, San Diego, Calif) was utilized for statistical analysis.
Pad2−/− improves survival in lethal HS
This was a lethal HS model as 100% of WT mice died within the first hour following HS (Fig. 1). In contrast, Pad2−/− mice had 0% mortality over the 7-day observation period after hemorrhage (P = 0.002).
Pad2−/− decreases infarction size and improves cardiac function after LAD ligation
After permanent LAD ligation, Pad2−/− animals had significantly reduced MI size on TTC staining compared with WT (60% vs. 75%, P < 0.05) (Fig. 2A). Pad2−/− exhibited improved cardiac function measured by EF and FS (P < 0.05) (Fig. 2B).
Pad2−/− increases β-catenin in heart tissue in HS
As shown in Figure 3, in the non-lethal HS model, β-catenin levels in heart tissue tended be higher in Pad2−/− mice compared with WT (sham) 12 h after hemorrhage. After HS, β-catenin levels were higher in Pad2−/− mice compared with WT (P = 0.04). Neither WT or Pad2−/− had β-catenin levels that exhibited significant differences compared after HS compared with sham.
Pad2−/− ameliorates acute lung injury in HS
In non-lethal HS, WT mice manifested ALI after 12 h. This was evident by hemorrhage, edema, and inflammatory cell infiltration. Pad2−/− mice displayed attenuated ALI. Both WT and Pad2−/− sham mice showed normal lung histology (Fig. 4A). ALI scores indicated there was a significant difference between WT mice and Pad2−/− mice subjected to HS (P = 0.03) (Fig. 4B).
Pad2−/− reduces CINC-1/KC level in serum in HS
In the non-lethal HS model, CINC-1/KC level in serum increased significantly at 12 h following HS in both WT group and Pad2−/− group. However, Pad2−/− animals had significantly lower elevation compared with the WT group (P = 0.01) (Fig. 5).
The present study is a continuation of our previous research which had shown that treatment with a novel PAD2/PAD4 inhibitor YW356 improved survival in a lethal HS rat model. The aim of this study was to determine if PAD2 inhibition was responsible for the pro-survival benefit in that model. We used PAD isoform specific knockout mice to achieve this aim. The survival experiment showed that knocking out PAD2 had a significant pro-survival effect in a lethal HS mouse model. To our knowledge, this is the first study showing specifically that loss of PAD2 function improves survival in HS. So far, the research on the role of PAD in hemorrhagic shock is limited. Other researchers have found that PAD4 activation exacerbates kidney ischemia-reperfusion injury and Pad4−/− mice are protected against kidney and liver injury after renal ischemia and reperfusion (6, 7). These results seem to be discordant with our findings. However, different models were used in these studies. The renal ischemia-reperfusion model is a single organ, non-lethal injury. In contrast, the HS model employed in this study created a lethal, global organ injury. It also involved exsanguination alone without subsequent resuscitation (unlike a classic ischemia-reperfusion insult). The role of PADs is likely to be organ and injury specific, which could help to explain differences observed between our study and others.
Early after massive hemorrhage, robust cardiac function is critical to maintain cardiac output and perfusion to end organs. Because of this, we chose to focus on the effect of Pad2−/− in the heart by employing a MI model using LAD ligation. Pad2−/− was importantly able to decrease MI size which was manifested by improved cardiac function compared with WT animals as measured by EF and FS. The decreased MI size demonstrates the protective effect of Pad2−/− against cardiac injury and cardiomyocyte cell death during ischemia. This could have a global protective effect to all other organs by improving EF and maintaining end organ perfusion. The cardioprotective effect of Pad2−/− is a very likely candidate to explain why Pad2−/− dramatically improved survival in HS.
The cardioprotection afforded by Pad2−/− was supported by β-catenin levels in the heart after HS. β-catenin is a downstream protein of Wnt/β-catenin signaling pathway which is involved in cell proliferation, migration, and other cell processes. The accumulation of β-catenin in cytosol drives it to translocate into nucleus and influence pro-survival gene activation and expression (14, 15). We have previously demonstrated that HS alters cardiac β-catenin levels (16). In this study, however, we did not demonstrate that either WT or Pad2−/− mice had significantly different β-catenin levels after HS compared with sham. Nonetheless, after HS Pad2−/− mice had significantly higher β-catenin levels than WT mice. This suggests that Pad2−/− upregulates the β-catenin pathway, which in turn could enhance cardiomyocyte survival. The relationship between PAD2 and β-catenin has been investigated by others as well. Targeted activation of PAD2 citrullinates β-catenin and increases β-catenin degradation, thereby blocking Wnt/β-catenin signaling (17).
Further, we focused on understanding systemic inflammation and end organ injury with knockout of Pad2 in HS. HS is regarded as a systemic inflammatory disease, where the excessive inflammatory cell recruitment and infiltration can lead to organ damage. To investigate this, we focused on ALI and CINC-1/KC. ALI and its more severe form, acute respiratory distress syndrome, can occur after hemorrhagic shock and has been shown to worsen the outcomes (18, 19). We discovered that Pad2−/− can attenuate the degree of ALI following HS. ALI is unlikely to be the etiology of mortality in this model, but it is an important contributor to morbidity and late mortality of survivors after an initial HS insult. We chose to analyze CINC-1/KC because it is known to contribute to systemic inflammation and induce neutrophil chemotaxis. CINC-1 is a rodent chemokine that belongs to the IL-8 family, and is homologous to the mouse keratinocyte-derived chemokine (KC) (20, 21). Our results showed that CINC-1/KC level in serum rose significantly in the WT mice after HS, and this was blunted to a significant degree in the Pad2−/− animals. As CINC-1/KC plays a key role in neutrophil recruitment to the organs, this may explain why Pad2−/− mice had decreased inflammatory cell infiltration, and less ALI on histological examination. These findings also fit with a more robust body of evidence showing the contribution of PAD to inflammatory conditions such as rheumatoid arthritis, multiple sclerosis, neurodegenerative diseases, and sepsis (22–24).
The present study has some limitations. Although the survival difference was statistically significant between the WT groups and Pad2−/− groups, the sample size was relatively small. However, with such a profound survival difference, ethical considerations limited us to increase the sample size. Additionally, we have a relatively limited analysis of end organ damage. The heart was chosen because of its importance for survival during and early after severe HS. The MI model does not perfectly reproduce the global hypovolemia state of hemorrhage. A hemorrhage event is likely a much more complicated event with hypoperfusion to coronary vessels with coincident poor filling of the ventricles, low stoke volume, and cardiac output. The MI model is targeted only at hypoperfusion to a discrete segment of the myocardium, which is clearly not as lethal of an insult given the animals were able to survive 24 h. The time analysis of the hearts to measure infarct size of 24 h is also a much different time than the early mortality seen within an hour for control animals after lethal hemorrhage. However, it is very unlikely that we would have seen differences in infarct size within an hour with TTC staining as others have shown it takes multiple hours to develop a stainable infarct with fixed ligation (25). Lung was chosen because development of ALI after HS impacts survival and can contribute to late mortality. Nonetheless, in future studies of HS it will be important to investigate the impact of Pad2−/− on other organs to understand if there are other organ specific effects. We measured CINC-1/KC as a marker of inflammatory cytokines and only did so at the 12-h time point. It would be ideal to test more cytokines at multiple time points to more fully understand the effect of Pad2−/− after HS on the inflammatory cascade. Finally, simply testing β-catenin levels in the heart is not a complete description of the molecular mechanism by which Pad2−/− improves survival in HS, and future studies will be required to more thoroughly examine these mechanisms.
In conclusion, this is the first study to identify the specific PAD isoform that plays a role in a HS model. We have discovered that elimination of PAD2 improves survival, attenuates markers of systemic inflammatory response and reduces ALI. PAD2 is a potential target for the development of targeted pharmacological treatments for HS and warrants ongoing investigation.
The authors thank Dr Scott Coonrod from Cornell University for the gift of the Pad2−/− mice.
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