Modulation of oxidative and nitrosative stress attenuates microvascular hyperpermeability in ovine model of Pseudomonas aeruginosa sepsis

In sepsis, microvascular hyperpermeability caused by oxidative/nitrosative stress (O&NS) plays an important role in tissue edema leading to multi-organ dysfunctions and increased mortality. We hypothesized that a novel compound R-107, a modulator of O&NS, effectively ameliorates the severity of microvascular hyperpermeability and preserves multi-organ function in ovine sepsis model. Sepsis was induced in twenty-two adult female Merino sheep by intravenous infusion of Pseudomonas aeruginosa (PA) (1 × 1010 CFUs). The animals were allocated into: 1) Control (n = 13): intramuscular injection (IM) of saline; and 2) Treatment (n = 9): IM of 50 mg/kg R-107. The treatment was given after the PA injection, and monitored for 24-h. R-107 treatment significantly reduced fluid requirement (15–24 h, P < 0.05), net fluid balance (9–24 h, P < 0.05), and water content in lung/heart/kidney (P = 0.02/0.04/0.01) compared to control. R-107 treatment significantly decreased lung injury score/modified sheep SOFA score at 24-h (P = 0.01/0.04), significantly lowered arterial lactate (21–24 h, P < 0.05), shed syndecan-1 (3–6 h, P < 0.05), interleukin-6 (6–12 h, P < 0.05) levels in plasma, and significantly attenuated lung tissue 3-nitrotyrosine and vascular endothelial growth factor-A expressions (P = 0.03/0.002) compared to control. There was no adverse effect in R-107 treatment. In conclusion, modulation of O&NS by R-107 reduced hyperpermeability markers and improved multi-organ function.


Results
Changes in systemic cardiopulmonary hemodynamics and biochemical variables during and after intravenous bacterial infusion. During the 60-min intravenous infusion of PA, body temperature (BT), heart rate (HR), mean arterial pressure (MAP), mean pulmonary artery pressure (mPAP), central venous pressure (CVP), systemic vascular resistance index (SVRI), pulmonary vascular resistance index (PVRI), and respiratory rate (RR) were increased, and cardiac index (CI) was decreased in all animals with the peak at 20-30 min after the start of the PA infusion. These changes returned to the baseline (BL) values by 90 min after the start of the bacterial infusion, with the exception of the BT, HR, mPAP, PVRI, and plasma lactate level, which gradually increased until 180 min after the initiation of the bacterial infusion. The greatest increases of endtidal CO 2 (EtCO 2 ) and CO 2 production (VCO 2 ) were observed at 30 min; these values then started decreasing 180 min after the start of the bacterial infusion. There was no significant difference in all variables between the two groups, indicating comparable injury in the two groups (Table 1).
Survival, qSOFA and mSOFA Scores, Plasma Lactate Clearance, and Lung Injury Score. During the 24-h study period, in both groups, MAP was decreased and BT, HR, mPAP, and CI were increased from their BL values, reflecting a state of hyperdynamic sepsis ( Table 2). The BT was significantly higher in R-107 treated sheep at 18-21 h (P < 0.05) compared to control. R-107 significantly attenuated the increases in LAP and CVP at 21-24 h (P < 0.01) compared to control. R-107 treatment also significantly decreased right ventricular stroke work index at 15 h (P < 0.05), and significantly attenuated the decreases in static lung compliance at 21 h (P < 0.05) as compared to control ( Table 2). The 24-h survival rate was 89% (8 out of 9 sheep) for the R-107 treatment group, and 69% (9 out of 13 sheep) for control (no statistical significance).
R-107 treatment significantly delayed the onset of sepsis compared to control, which was confirmed by the time that sheep met quick SOFA (qSOFA) criteria. Six of 9 sheep in the R-107 group met the qSOFA score criteria within 6.8 ± 2.0 h, whereas all 13 sheep in the control group met these criteria within 3.3 ± 0.7 h from the start of the PA infusion (P = 0.04) (Fig. 1A). www.nature.com/scientificreports/ R-107 treatment significantly attenuated the increases in the modified sheep SOFA (mSOFA) score at 24-h in the survived animals (n = 8 in the R-107 treatment group and n = 9 in control, 2.9 ± 1.0 vs. 5.9 ± 1.0, P = 0.04) as compared to control (Fig. 1B). The measured variables of mSOFA score in both groups are shown in Table 3.
Also, the lung injury score (LIS) at 24-h in the survived animals was significantly lower in the R-107 treatment group as compared to control (n = 8 in the R-107 treatment group and n = 9 in control, 1.5 ± 0.2 vs. 2.2 ± 0.2, P = 0.01) (Fig. 1C).
Fluid balance, hematocrit changes, plasma protein concentration, and total amount of pleural and ascitic fluid at necropsy. The fluid requirement was significantly lower in the R-107 treated group at 15-24 h (P < 0.05) ( Fig. 2A), and cumulative urine output at 15-24 h (P < 0.05) (Fig. 2B) was significantly higher in the R-107 treated group compared to control. The net fluid balance was significantly attenuated in the R-107 treated group as compared to control at 9-24 h (P < 0.05) (Fig. 2C). The hematocrit levels were comparable between the treated and control groups.
The plasma protein concentration was significantly higher in the R-107 treated group compared to control at 21-24 h (P < 0.05) (Fig. 2D).
The combined volume of pleural and ascitic fluid at euthanasia was significantly lower in the R-107 treated group as compared to control (21.1 ± 6.2 vs. 57.7 ± 4.7 mL/kg BW, P < 0.001) (Fig. 2E).
Organ extravascular water content and bacterial clearance. The organ extravascular water content was determined by measuring organ wet-to-dry weight ratio (W/D) at euthanasia. The R-107 treated group displayed significantly lower water content in the lung, heart, and kidney compared to control (lung: 7.5 ± 0.  www.nature.com/scientificreports/ Oxidative and nitrosative stress, glycocalyx, and inflammatory mediators. The modulation of O&NS by the R-107 treatment was measured by the levels of 3-nitrotyrosine in the lung tissue. The 3-nitrotyrosine levels in lung tissue homogenate collected at the euthanasia was significantly lower in the R-107 treated group compared to control (0.07 ± 0.01 vs. 0.13 ± 0.02, P = 0.02) (Fig. 4A). R-107 significantly decreased plasma levels of shed syndecan-1 (Sdc1) at 3 and 6 h (37.0 ± 6.6 vs. 88.9 ± 13.4 and 26.7 ± 7.7 vs. 68.8 ± 10.9, P = 0.005 and 0.03, at 3 and 6 h, respectively) compared to control (Fig. 4B).

Discussion
In this study, we demonstrated that modulation of O&NS by R-107 significantly reduced microvascular hyperpermeability and body fluid retention, resulting in improved multi-organ function and survival in septic sheep. Previously, we demonstrated that the modulation of O&NS by R-100 significantly reduces systemic fluid retention and improves pulmonary gas exchange in the model of ovine pneumonia/sepsis 21 . The present study results were in an agreement with previous work in terms of reduced fluid retention, organ edema, and microvascular hyperpermeability. The survival rate in control groups in present and previous studies was also comparable (69% vs. 65-70%, respectively 23,37 . In the present study, we further showed that R-107 treatment improved bacterial clearance in the lung and kidney, inhibited increases in inflammatory mediators (plasma shed Sdc1, IL-6, and lung VEGF-A levels), delayed the onset of sepsis (measured by the qSOFA score), attenuated multi-organ dysfunction (measured by the mSOFA score), and improved multi-organ function. In ICU patients with sepsis and septic shock, positive fluid balance, at least in part, due to this microvascular hyperpermeability is not only the independent predictor of mortality, but it also is linked to the increased ICU-days, ventilator-days, and total hospital care costs [38][39][40][41][42] . Therefore, therapies targeting microvascular hyperpermeability and edema formation during sepsis are of particular interest.
As mentioned, sepsis increases microvascular hyperpermeability and excessive fluid retention and causes multiple organ dysfunction by excessive O&NS 7-9,43,44 . Excessive O&NS causes endothelial cell damage and endothelial glycocalyx layer disruption [43][44][45][46] . The endothelial glycocalyx layer controls endothelial permeability to water and serves as a barrier to neutrophil/bacteria migration into organ interstitial space from the bloodstream 7-9,44-47 . The Sdc1 is one of the major components of the glycocalyx, and the levels of shed Sdc1 in plasma are considered   49,50 .
In addition, VEGF-A, a well-known inflammatory mediator and potent vascular hyperpermeability factor, is increased in septic conditions 33,35,51 . In our previous studies, we have shown increases in lung tissue VEGF and myeloperoxidase activity associated with sever microvascular hyperpermeability 35 .
The results of the present study demonstrated that R-107 treatment significantly reduced the degree of O&NS as evidenced by increases in lung tissue 3-nitrotyrosine levels. We also showed that R-107 significantly reduced  www.nature.com/scientificreports/ the plasma levels of shed Sdc1 and IL-6 and significantly attenuated VEGF-A levels in lung tissue compared to control. Although, these findings do not represent causation, we speculate that R-107 attenuated microvascular permeability in this model by inhibiting the potent permeability factor VEGF and preserving endothelial glycocalyx through modulation of O&NS stress.
In the present study, we reported that the number of bacteria in lung and kidney tissue were significantly lower in the R-107 treatment group compared to control. The underlying mechanisms for this finding are not fully understood. Lubkin et al. reported that PA secretes toxins that disrupt the endothelial cell monolayer barrier mechanism via the induction of oxidative stress 52,53 . We speculate that protection of the endothelial barrier by R-107 may be attributed to the reduced number of bacteria in organ tissues. Of note, circulating numbers of neutrophils were not affected by R-107, indicating that differences in bacterial numbers between the two groups were not related to variations in the number of neutrophils.
Further, increases in plasma shed Sdc1 in control were unlikely to have been impacted by the volume of resuscitation fluid because the hematocrit, plasma protein concentration, and hemodynamic variables were comparable in both groups at 3-6 h, when the plasma shed Sdc1 levels were significantly lower in the R-107 treated group compared to control.
Our present study has several limitations. First, we did not show causative factors by which R-107 reduced microvascular hyperpermeability, rather we showed co-association of the numbers of potent inflammatory mediators with increased vascular permeability. Second, we did not use the supportive therapies, such as antibiotics and vasopressors, which are the standard therapies for sepsis. However, we aimed to explore a "pure" effects of testing compound without any drug interactions. Third, the study period was relatively short (24-h), which does not consider the concomitant diseases or factors that are associated with human sepsis. However, the model allowed us to most closely mimic the human hyperdynamic sepsis, continuously monitoring cardiopulmonary hemodynamics in a conscious state without the effects of anesthetics. Finally, we have compared variables only in two groups of septic sheep (treated and control) without including the variables obtained from uninjured healthy (Sham) sheep. However, we have repeatedly reported in our previous studies that the preceded surgical procedures in the Sham sheep did not affect variables reflecting multiorgan functions (i.e., cardiopulmonary hemodynamics, pulmonary function) and survival 23,37 .
In conclusion, modulating excessive O&NS stress by R-107 may be considered as an effective and safe therapeutic option for management of sepsis-induced microvascular hyperpermeability. Future studies are warranted to further investigate the mechanisms of how moderation of O&NS by this potential therapeutic compound "R-107" attenuates the pathophysiology of sepsis-induced microvascular hyperpermeability.  www.nature.com/scientificreports/ Twenty-two adult female Merino sheep (body weight [BW] 36.8 ± 1.0 kg) were used. Briefly, animals were anesthetized with an intravenous injection of ketamine and isoflurane inhalation, and multiple vascular catheters were surgically inserted (Swan-Ganz, femoral arterial, and left atrial catheters). Pre-and post-surgical analgesia was provided with long-acting (for 72 h) Buprenorphine SR™ (0.05 mg/kg, SR Veterinary Technologies, Windsor, CO). Merino sheep were chosen because of their close resemblance of the pathophysiologic and immune responses to infection that are seen in humans [25][26][27] .

Methods
After 5-7 days following instrumentation, BL cardiopulmonary hemodynamic variables were collected (Table 2), as previously described 23,24 . After the BL data were collected, a tracheostomy tube and urine catheter were inserted under ketamine and inhaled isoflurane anesthesia, and animals were placed on a mechanical ventilator (AVEA; Carefusion, Yorba Linda, CA) with the initial settings of a pressure-regulated volume control assistcontrol mode, tidal volume (TV) of 12 mL/kg, positive end-expiratory pressure of 5 cmH 2 O, RR of 20 breaths/ minute, and inspired oxygen concentration (FiO 2 ) of 0.21. Then, 1.0 × 10 10 CFUs of Pseudomonas aeruginosa (strain; PD-05144 [12-4-4, BRK-1244, NCIB-10780, NRRL-B-3224], catalog #: ATCC® 27,317™, ATCC, Manassas, VA) suspended in 50 mL of warm 0.9% sodium chloride were intravenously injected (IV) via the jugular vein over 60 min in a conscious state. The variables of systemic hemodynamics were continuously monitored during and until 180 min after the initiation of the bacterial IV infusion. (Table 1). Arterial lactate levels were also determined during this time period.
Animal grouping, drug treatment, and post-injury care. After the injury, animals were randomly allocated into two groups: (1) control: treated with an intramuscular injection (IM) of saline, n = 13; and 2) R-107: administered with IM 50 mg/kg R-107, n = 9. The R-107 was injected into the animal's right quadriceps immediately after completing the infusion of bacteria.
Briefly, R-107 has a multi-functional prodrug technology to target redox imbalance of O&NS. R-107 is a prodrug ester that hydrolyzes to form R-100 21 , a molecule serving as: a NO donor via its organic nitrate, and a broad-spectrum catalyst of O&NS degradation via its nitroxide moiety (hydroxymethylproxyl). The spectrum of O&NS degradation of R-100 includes: O 2 dismutation, catalase-like activity (detoxifying H 2 O 2 ), and peroxynitrite decomposition. There are at present no approved agents with this multi-functional action. R-107 was provided from the Salzman Group Inc. (Beverly, MA).
Animals were fluid resuscitated with lactated Ringer's solution (LR; Baxter Healthcare Corporation, Deerfield, IL), starting with an initial infusion rate of 2 mL/kg/hr for 3 h. Thereafter, the LR rate was adjusted every 3 h to maintain hematocrit close to the BL levels ± 3%. Fluid input and urine output were monitored hourly and cumulative fluid balance was calculated, as previously described 23,24 . Quick SOFA and Modified Sheep SOFA Score. In order to assess the onset of sepsis and the severity of multi-organ dysfunction during sepsis, we used qSOFA and mSOFA scores, as previously described 1,24,29 . We measured the time to meet the qSOFA criteria from the initiation of the PA injury. Also, the mSOFA score included the values of animal neurological status, MAP, PaO 2 /FiO 2 ratio, total platelet counts measured by ADVIA-120 (Siemens Healthcare Diagnostics, Deerfield, IL), and plasma total bilirubin and creatinine concentrations measured by the hospital clinical chemistry laboratory. The neurological status of animal was assessed by the Simplified Sheep Neurological/Alertness Assessment score 29 . The qSOFA score was measured hourly until the animals met the criteria, and the mSOFA score was measured at BL and at the 24-h timepoint.
Euthanasia, tissue collection, and tissue extravascular water content analysis. After completion of the 24-h study period, animals were euthanized with injection of ketamine (40 mg/kg), buprenorphine (0.01 mg/kg), and xylazine (3.0 mg/kg), following the IACUC approved protocol and American Veterinary Medical Association Guidelines for Euthanasia 30 . Immediately after euthanasia, organs and tissues were collected, and lung, heart, and kidney tissue water content were measured by W/D, as previously described 23,24,31 . Bacterial clearance in lung and kidney. To assess bacterial clearance in the lung and kidney, a 1.0-g section of the dorsal edge of right lung middle lobe and the kidney cortex were taken at necropsy and homogenized in 2 mL of 1 × phosphate-buffered saline, using a Bullet Blender (Next Advance, Averill Park, NY) following the device protocols (https:// www. nexta dvance. com/ homog enizer-tissue-cell-cultu re-bullet-blend er-suppo rt/ homog enizer-cell-disru pter-proto cols/). Then, 200 µL of the tissue homogenates were transferred onto soy agar plates. The plates were incubated for 24 h at 37 °C for bacterial CFUs counts, as previously described 29,32 . Western blotting and enzyme-linked immunosorbent assay. Levels of lung tissue 3-nitrotyrosine (06-284; MilliporeSigma, MA) and VEGF-A (ab46154; Abcam, MA), which is a major mediator of microvascular hyperpermeability [33][34][35] , were determined by automated capillary Western blot analysis (Wes™ ; ProteinSimple, San Jose, CA), as previously described 24