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Background/Aim: Circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. The aim of this study was to implement a simple, yet effective quantitative method for measuring the cfDNA in serum and to investigate the relationship between cfDNA and the occurrence of recurrence in breast cancer (BrCa) patients. Patients and Methods: A total of 240 cases were selected, which comprised different subtypes of BrCa patients and control individuals. We selected 20 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. SYBR green was used as a reporter molecule to estimate the amount of cfDNA in these serum samples. Results: A global Wilcoxon analysis was performed to compare the cfDNA abundance between non-recurrent and recurrent patients. The amount of cfDNA was higher in recurrent patients (recurrent vs. non-recurrent ratio=1.3; p=0.03; AUC=0.76) compared to non-recurrent patients. The data between normal/healthy controls and non-recurrent patients indicated no significant differences (n=20 in each group, healthy to non-recurrent ratio=1.03; p=0.20; AUC=0.61). Conclusion: We implemented a straightforward one-step technique to measure the amount of cfDNA in serum, which can translate into a clinical diagnostic tool in the near future. The high levels of cfDNA in the serum of recurrent BrCa patients compared to non-recurrent BrCa patients indicates a possible uncovered role for circulating genetic information, which either contributes to the cancer recurrence phenomenon or at the very least, serves as an identifier for the potential of recurrence.

COVID-19 is a global issue, with over 6.25 million cases in 213 countries and territories on June 1, 2020. Although this virus infects all groups, data indicate that the risk for severe disease and death is much higher in older men, which coincides with the same group of patients at risk for prostate cancer. A recent Italian study investigated the prevalence and severity of COVID-19 in men with prostate cancer. This study indicated that of a total of 4,532 men with COVID-19, from the Veneto region of Italy, 9.5% (n=430) had cancer and out of those around 30% (n=118) had prostate cancer. Data also indicated that male cancer patients had a 1.8-fold increased risk of COVID-19 infection and developed a more severe disease. Interestingly, they observed that the prostate cancer patients (n=4) treated with androgen-deprivation therapy (ADT) were less likely to develop COVID-19, and in those who were infected, the disease was less severe. In this current study, we focused on determining the genetic basis of the higher COVID-19 prevalence and severity in male patients and particularly for prostate cancer patients. Researchers found two genes that are essential for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). ACE2 is a SARS-CoV-2 receptor, whereas the serine protease, TMPRSS2, primes the virus for cell entry through cleavage of the viral spike protein (S). The expression of TMPRSS2 is significantly high in normal prostate tissue and is regulated in large part by an androgen response element in the promoter region. Therefore, we decided to investigate the status of these two genes in various tumors from The Cancer Genome Atlas (TCGA) database using the cBioportal platform. We analyzed over 46,000 tumor samples from 176 studies and found that aggressive metastatic prostate cancer, including neuroendocrine prostate cancer (NEPC), has significantly higher amplification (copy number alteration) of the ACE2 and TMPRSS2 genes compared to other cancers. Next, we focused on drugs that could simultaneously target ACE2 or TMPRSS2 and oncogenic pathways and would be beneficial for prostate cancer patients infected with SARS-CoV-2. Although several inhibitors are validated in literature for both ACE2 and TMPRSS2, very limited studies were performed to see the effect on cancer cells. Therefore, we analyzed a cytotoxic effect database of over 130,000 drugs on NCI-60 cell lines with COMPARE algorithm and found two relevant compounds, NSC-148958 (FT-701) and NSC-280594 (triciribine phosphate), which target ACE2 and TMPRSS2, respectively. Computational data are currently validating different prostate cancer cell-lines and their response to these drugs. In summary, our findings provide the premise that men who are at risk for or diagnosed with prostate cancer may be more susceptible to severe infection and death in response to SARS-CoV-2 due to the high expression of ACE2 and TMPRSS2, and triciribine phosphate and FT-701 could be a therapeutic intervention to target co-occurrence of COVID-19 and prostate cancer. Citation Format: Alakesh Bera, Eric Russ, Digonto Chatterjee, John Karaian, Madhan Subramanian, Sreejato Chatterjee, Surya Radhakrishnan, Michael Eklund, Harvey Pollard, Meera Srivastava. A prediction of prostate cancer deaths spiking by SARS-CoV-2 infection [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S01-03.

Introduction: Biopsy of the allograft is the gold standard for assessing kidney allograft dysfunction. The aim of our pilot study was to identify serum biomarkers that could obviate the need for biopsy. Materials and Methods: We conducted a study to identify the biomarkers in the serum from different groups of chronic kidney disease (CKD) patients and kidney transplanted patients vs. healthy individuals. The four groups (n=25 in each group) were as follows: 1) Patients with unstable kidney allograft transplants requiring biopsy for cause, 2) Patients with stable kidney allograft transplants, 3) Patients with CKD not on immunosuppressive therapy and, 4) healthy subjects. We measured the activity and level of serum alkaline phosphatase (ALP) and other liver enzymes (alanine transaminase (ALT) and aspartate transaminase (AST)) as potential serum biomarkers in acute allograft dysfunction. Results: We found that ALP correlated with allograft biopsy findings, liver function, and clinical outcomes and possibly graft survival. Additionally, AST and ALT were higher in patients with graft rejection compared to non-rejected and stable kidney transplants. Moreover, the low Pearson correlations (r- values) between ALP level with age (r=0.179), gender, body mass index (r=0.236), creatinine (r=0.044) or estimated glomerular filtration rate (r=0.048) suggest that ALP may be an independent biomarker which is relatively unaffected by other individual-level variables. Conclusion: ALP may be a putative biomarker to predict kidney allograft function and rejection. Data also indicated that liver function plays an important role for the overall success of kidney transplantation.

Breast Cancer is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Identification of new biomarkers could be key for early diagnosis and detection. Vitronectin, a glycoprotein that is abundantly found in serum, extracellular matrix, and bone, binds to integrin αvβ3, and promotes cell adhesion and migration. Current studies indicate that patients with amplified vitronectin levels have lower survival rates than patients without amplified vitronectin levels. In this study, we focused on the role of vitronectin in breast cancer survival and its functional role as a non-invasive biomarker for early stage and stage specific breast cancer detection. To confirm that the expression of vitronectin is amplified in breast cancer, a total of 240 serum samples (n = 240), 200 from breast cancer patients and 40 controls were analyzed using the Reverse Phase Protein Array (RPPA) technique. Of the 240 samples, 120 samples were of African American (AA) descent, while the other 120 were of White American (WA) descent. Data indicated that there were some possible racial disparities in vitronectin levels and, differences also seen in the recurrent patient samples. Next, we tried to uncover the underlying mechanism which plays a critical role in vitronectin expression. The cellular data from four different breast cancer cell lines- MCF7, MDA-MB-231, MDA-MB-468, and HCC1599 indicated that the PI3K/AKT axis is modulating the expression of vitronectin. We believe that vitronectin concentration levels are involved and connected to the metastasis of breast cancer in certain patients, specifically based on recurrence or ethnicity, which is detrimental for poor prognosis. Therefore, in this current study we showed that the serum vitronectin levels could be an early marker for the breast cancer survival and we also determine the cellular signaling factors which modulate the expression and concentration of vitronectin.

The chromosome 8q24 gene contains functional risk loci for multiple cancers, including prostate, colon, and breast. Breast cancer (BrCa) is one of the deadliest diseases among women. Our previous studies using TCGA clinical survival data (Cell. 2018; 173(2):400-416.e11) indicated that several genes are contributed significantly towards the survival of patients with BrCa. Over 75 such most significant genes were screened for further studies in terms of their contribution towards BrCa patient's survival. We found two genes (EXOSC4 and VPS28) with over 10% genetic copy number alterations located at chromosome 8q24 gene loci. Exosome Component 4 (EXOSC4) is located at 8q24.3 and associated with a non-catalytic component of the RNA exosome complex, which has 3' to 5' exoribonuclease activity. It also participates in cellular RNA processing and degradation events. However, the functional role of EXOSC4 towards BrCa progression or survival remains unknown. To understand the molecular basis of an elevated level of EXOSC4 in BrCa propagation, we screened the expression of this protein in different BrCa cell-lines. We also compared the expression of VPS28 within same cell lines. Data indicated that EXOSC4 was co-expressed with VPS28 and Myc, and was also secreted into extracellular space. Therefore, we next evaluated the level of EXOSC4 in patient's serum samples by reverse phase protein array (RPPA). We have well defined and categorized BrCa patients serum samples (n=240) from the Clinical Breast Care Project. The data indicated that the elevated level of EXOSC4 is associated with worse overall survival. In addition, results also showed that the high level of EXOSC4 is linked with breast cancer recurrence.In summary, through multiple modality studies we have confirmed the copy number alteration of EXOSC4 is associated with BrCa patient survival. Once validated, serum level of EXOSC4 could be a predictive biomarker for BrCa progression and survival. Additional experiments are ongoing to evaluate the regulatory role of the EXOSC4 gene which has the potential to develop into a novel therapeutic intervention of BrCa. Funding Sources: This work was supported by the grant to Dr. Meera Srivastava (DoD, DAMD17-03-1-0107). The work was also supported by the Collaborative Health Initiative Research Program (CHIRP) funding (CHIRP ID# IAA-A-HL-14-007). Disclaimers: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of The Uniformed Services University of the Health Sciences (USUHS), The Henry M. Jackson Foundation (HJF), the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. Correspondence: Email: meera.srivastava@usuhs.edu Citation Format: Alakesh Bera, John Karaian, Madhan Subramanian, Michael Eklund, Eric Russ, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Meera Srivastava. EXOSC4, a novel gene at chromosome 8q24 loci is linked with breast cancer progression and is a prognostic marker for breast cancer survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4322.

Transforming growth factor β (TGFβ) is one of the most important signaling pathways associated with cellular proliferation, and its dysregulation can lead to tumor development. TGFβ can act as either a tumor suppressor or a tumor promoter depending upon the cellular context and genetic alterations. TGFβ receptor type-2 (TGFβR2) is the ligand-binding receptor, and data shows that the aberration of TGFβR2 is associated with many different cancers including colon, gastric, biliary, pulmonary, and ovarian cancers. In this study, we focused on uncovering the functional role of TGFβR2 in regulating breast cancer (BrCa) progression. First, we analyzed the genomic alterations of TGFβR2 in a sample set from The Cancer Genome Atlas (TCGA, n=1084) on the cBioprotal platform. We focused on obtaining genetic modifications, including mutations and copy number alteration. Data indicated that almost 25% of the patient’s samples having TGFβ amplified copy number; however, only less than one percent patients have amplified copy of TGFβR2 and other TGFβ receptors, within across this sample set. Next, we have screened the level of TGFβR2 protein expression in different BrCa cell-lines. We found that the elevated level of TGFβR2 is associated with the most aggressive triple-negative MDA-MB-231 cells. It is also found that the TGFβR2 is secreted into extracellular space. Therefore, we next evaluated the expression level of TGFβR2 in patient’s serum samples. We have well defined and categorized BrCa patients serum samples (n=240) from the Clinical Breast Care Project led by Walter Reed National Military Medical Center, Bethesda. The differential expression level of TGFβR2 in serum indicated that the elevated level of TGFβR2 is associated with BrCa patients compared with healthy individuals. Furthermore, the serum level of TGFβR2 is higher in the African American patient population when compared to Caucasian patients, and most significant differences were found within Luminal B1 sub-type BrCa cases. To better understand the mechanism of the higher level of TGFβR2 protein expression, in association with aggressiveness, we hypothesized that increased levels of TGFβ ligand are inducing the expression of the receptor (TGFβR2) and eventually stimulating the oncogenic pathway. We have used a series of BrCa cell lines and incubated them with different concentrations of TGFβ ligand. The level of TGFβR2 protein was increased in BrCa cell lines after TGFβ ligand treatment. Increased levels of TGFβ2 ligand induces the expression of the relevant receptor. In summary, we quantified the expression levels of TGFβR2 in patients’ serum samples, which can be used to create a set of stage and race-specific candidate protein biomarkers for BrCa. We also found that the increased level of TGFβ ligand-induced the expression of the TGFβR2 receptor. We can also conclude that TGFβR2 is an important biomarker and possibly plays a role in racial disparities in breast cancer progression. Citation Format: Alakesh Bera, John Karaian, Harvey B Pollard, Hai Hu, Craig D Shriver, Meera Srivastava. TGF-beta receptor type-2 expression regulates breast cancer progression and is a prognostic marker for racial disparities [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A073.

Background: Quantitative estimation of circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. However, a simple, straightforward technique is unavailable to estimate the cfDNA in clinical labs. Moreover, the prognostic value of cfDNA in patients with breast cancer (BrCa) is currently under debate. The aim of this study was to develop a simple yet effective quantitative method for measuring the cfDNA in serum and to eventually investigate the relationship between cfDNA and the occurrence of recurrence in BrCa patients. Methods: A total of 240 patient cases (n=240) were selected and are comprised of different subtypes of breast cancer patients and control individuals. We selected 21 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. For the compare studies, each of the recurrent and non-recurrent serum samples was incubated with the SYBR Green I (2 μM). A standard graph was also made with known DNA concentration to calculate the amount of cfDNA in these recurrent and non-recurrent serum samples. Additionally, a comparative study was also performed with the serum of patients with non-recurrent BrCa versus healthy patients. Results: We develop a simple fluorescent based measuring technique which can easily estimate the cfDNA in one step. SYBR Green binds to DNA, and as a result, the fluorescence of SYBR Green increases substantially. Global Wilcoxon analyses were performed to compare the cfDNA amount between non-recurrent and recurrent patients. There is a significant difference in fluorescent intensities between recurrent patients' samples versus non-recurrent patients which are directly proportional to the cfDNA levels. The amount of cfDNA is higher in recurrent patient (ratio is 1.3 up; p= 0.03; AUC=0.76) compared to similar non-recurrent patients. While we compared the fluorescence data between normal/healthy patients versus non-recurrent is turned out as non-significant (healthy to non-recurrent ratio = 1.03; p= 0.20, AUC=0.61). Conclusion: In this current study, we developed a straightforward one-step technique to measure the amount of cfDNA in serum, which can easily translate into a clinical diagnostic tool. To the best of our knowledge, this is the first report which demonstrates serum cfDNA as an early detection marker for recurrent breast cancer patients. The relatively high level of cfDNA in the serum of recurrent breast cancer patients compared to non-recurrent breast cancer patients indicates an uncovered circulating genetic information which triggers the cancer recurrence pathway to relapse cancer in the near future. Citation Format: Bera A, Eidelman O, Russ E, Landa A, Karaian J, Eklund M, Hu H, Pollard HB, Shriver CD, Srivastava M. Circulating cell-free DNA in serum as a marker for the early detection of tumor recurrence in breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-26.

Objectives: To evaluate the effectiveness of normal saline, hypertonic saline, and Ringer's lactate solution followed by blood infusion in ameliorating the physiological, biochemical, and organ functions following hemorrhagic shock (HS) in rats. Materials and Methods: Anesthetized, male Sprague-Dawley rats underwent computer-controlled HS, and were randomly divided into five groups consisting of (1) sham, (2) HS without resuscitation, (3) resuscitation with normal saline, (4) resuscitation with hypertonic saline, and (5) resuscitation with Ringer's lactate solution. All resuscitated animals were infused with subsequent infusion of shed blood. Animals were continuously monitored for physiological, hemodynamic, biochemical parameters, and organ dysfunctions. Results: Non-resuscitated animals were unable to survive due to hypotension, poor oxygen metabolism, and lactic acidosis. Although these HS related parameters were corrected by all the fluids used in this study, additional blood infusion was more effective than fluid resuscitation alone. Also, hypertonic saline was more effective than Ringer's lactate solution, and normal saline was the least effective in preserving the liver and kidney functions and muscle damage. Conclusions: All crystalloid fluids were significantly more effective in reversing the HS outcome when used with blood infusion, but hypertonic salinewith blood was more effective in preventing the organ damage than Lactated Ringers solutions or normal saline in the treatment of HS.

Background Skeletal muscle fibers from malignant hyperthermia (MH)-susceptible humans and swine are markedly more sensitive to ryanodine receptor (RyR1) agonists than those from normal individuals. Reproducible shifts in the dose-response of skeletal muscle to caffeine and halothane are the basis of the current in vitro diagnostic caffeine-halothane contracture test. In an attempt to develop a less invasive MH diagnostic test, the authors determined the effects of RyR1 agonists (caffeine, 4-chloro-m-cresol [4CmC], and halothane) on the adductor muscle with respect to the lactate-pyruvate (L/P) system that was percutaneously dialyzed using a microdialysis technique in homozygous MH-susceptible compared with normal swine. Methods Animals were anesthetized (ketamine-propofol) and artificially ventilated. Sets of six CMA/20 microdialysis catheters were implanted; each catheter was perfused with different RyR1 agonist concentrations. After a 30-min equilibration after implantation, one of the catheters was perfused (2 microl/min) with vehicle (0.9% saline or lipid emulsion), and the other five were perfused with caffeine (1-64 mM), 4CmC (0.1-8 mM), or halothane (prepared in lipid emulsion; 10-500 mM). Outflow dialysate fractions collected at 10-min intervals and L/P parameters were measured enzymatically. Results Only in the MH-susceptible group did all RyR1 agonists increase dialysate L/P in a dose-dependent manner. The dose-effect relations were most prominent with 4CmC. With the halothane lipid emulsion, data scatter was high compared with that of the caffeine group and especially the 4CmC group. There were no signs of global muscle rigidity, systemic hypermetabolism, or a clinical MH episode during microdialysis RyR1 perfusion. Conclusions The authors data demonstrate that the in vivo muscle microdialysis of the porcine L/P system reveals distinct differences between MH-susceptible and MH-normal muscle, especially in response to highly specific RyR1 agonists such as 4CmC. The microdialysis L/P technique seems to have an MH diagnostic potential in the clinical setting.

Background: Dysfunctional mitochondria have been widely accepted as one of the key targets and a mediator of secondary cell injury and organ failure during hemorrhagic shock (HS). The liver is known to be the first organ to display the signs of injury during HS. This report describes experiments to determine whether modulation of hepatic mitochondrial dysfunctions by pharmacologic agents could prevent liver injury in rats subjected to HS. Methods: In this study, Sprague-Dawley rats were either treated as controls or subjected to computer-controlled arterial hemorrhage (40 mmHg) for 60 min followed by resuscitation with hypertonic saline, hypertonic -hydroxybutyrate, or hypertonic sodium pyruvate for the next 60 min before death. During the course of the experiment, animals were continuously monitored for hemodynamic and metabolic parameters. At the end of the experiment, the liver was excised and examined for oxidative injury, mitochondrial functions, expression of nitric oxide synthase, and indicators of apoptosis. Results: In comparison to hypertonic saline and hypertonic -hydroxybutyrate, pyruvate significantly protected the liver from oxidative injury, prevented the up-regulation of nitric oxide synthase, inhibited pyruvate dehydrogenase deactivation, and improved cellular energy charge and mitochondrial functions. In addition, pyruvate also reduced cleavage of poly‐ adenosine diphosphate ribose polymerase by preventing leakage of mitochondrial cytochrome c in the liver of HS animals. Conclusions: These data suggest that modulation of mitochondrial metabolic functions is likely to be one of the important mechanisms by which pyruvate exerts its protective effects on the liver during HS and resuscitation in rats. MULTIPLE organ failure in hemorrhagic shock (HS) has been commonly related to metabolic failure due to dysfunctional mitochondria. The liver is known to be the first organ to display the signs of injury during HS due to its high mitochondrial content, increased metabolic rate, lactic acid clearance, and increased production of oxygen and nitrogen species in comparison with other organs. 1–3 A relation between mitochondrial dysfunction and the irreversibility of pathologic damage in liver has been reported. 4–6 Therefore, it is logical to hypothesize that the pharmacologic agents that can reduce reactive oxygen and nitrogen species and prevent mitochondrial damage may offer a promising treatment for HS. To test our hypothesis, we examined and compared the effects of pyruvate with another pharmacologic agent, -hydroxybutyrate (-HOB), in resuscitative fluids of rats subjected to HS. The rationale for the use of pyruvate and -HOB is that they are antioxidants, decrease membrane permeability, and produce acetyl coenzyme A as the immediate substrate of the Krebs cycle to generate mitochondrial matrix reduced adenine dinucleotide for oxidative phosphorylation. 7 The uptake of -HOB by mitochondria is adenosine triphosphate (ATP) dependent, whereas pyruvate requires the activity of the mitochondrial enzyme pyruvate dehydrogenase, which is regulated by the content of its E-1 subunit. 8 However, it is not known whether pharmacologic activation of pyruvate dehydrogenase complex (PDH) and up-regulation of its E-1 subunit could be linked to the improved hepatococellular ATP, mitochondrial functions, and decreased apoptosis. In addition, the use of -HOB in the treatment of HS has not been extensively studied in HS. On the other hand, recent work from our laboratory and those of others have shown the use of pyruvate administration in restoring the cellular metabolic functions in the brain and heart and also in enhancing the survival rates during HS or ischemia by preventing the loss of ATP. 7,9 –12 Despite this, the mechanisms for these responses are not fully understood, and the role of pyruvate in restoring the mitochondrial energetic functions in one of the most important organs, the liver, is still unresolved. The use of pyruvate and -HOB as alternate adjuvants to metabolic resuscitative fluids may therefore provide a rational approach for the development of an effective metabolic therapy for HS by maintaining the hepatocellular functions.

Background To determine the role of nitric oxide and adenosine triphosphate-sensitive potassium (KATP) vascular channels in vascular decompensation during controlled hemorrhagic shock in swine. Methods Thirty instrumented, anesthetized adolescent Yorkshire swine were subjected to controlled isobaric hemorrhage to a mean arterial pressure of 40 mmHg for 2 h (n = 6) or 4 h (n = 10) or 50 mmHg for 4 h (n = 8). An additional six animals were used as anesthetized instrumented time controls. During controlled hemorrhage, plasma and tissue samples were obtained every 30 to 60 min. Before euthanasia, tissue (carotid artery, lung, liver, and aorta) was obtained for analysis of nitrate concentrations and nitric oxide synthase activity. Isolated carotid artery ring reactivity to norepinephrine was also determined with and without glibenclamide. Results Animals hemorrhaged to 40 mmHg decompensated earlier than animals hemorrhaged to 50 mmHg. Plasma nitrate concentrations and nitric oxide synthase activity rose consistently throughout hemorrhage in both groups. However, they were substantially higher in the mean arterial pressure 40 group. Constitutive nitric oxide synthase activity was the major contributor to total nitric oxide synthase activity throughout the protocol with only the animals maintained at 40 mmHg for 4 h showing evidence of inducible nitric oxide synthase activity. Profound KATP channel activation and hyporeactivity of isolated vessel rings to norepinephrine was not observed until 4 h after the initiation of hemorrhagic shock. Only those animals with inducible nitric oxide synthase activity showed a decreased response to norepinephrine, and this hyporeactivity was reversed with the KATP channel inhibitor, glibenclamide. Conclusions The data indicate that profound KATP activation associated with increased nitric oxide concentrations and inducible nitric oxide synthase induction is a key factor in vascular smooth muscle hyporeactivity characteristic of the late decompensatory phase of hemorrhagic shock in swine.

The inadequate availability of fuel substrates and sharp decline in cellular ATP have been implicated in a cascade of events associated with cell death and organ failure during hemorrhagic shock (HS). In this in vivo swine model of severe prolonged HS, the effect of exogenous pyruvate administration on various markers of cell damage in brain and liver was examined. Thirty minutes after the start of controlled arterial hemorrhage, 30% sodium pyruvate, 10% saline, or 0.9% saline was administered via jugular vein. Four hours after the initiation of hemorrhage, tissue samples from brain and liver were obtained and examined for the cellular and molecular markers of cellular damage. Results of our study suggest that pyruvate prevents loss of total NAD content, cleavage of poly-ADP ribose polymerase (PARP), and inhibits lipid peroxidation in both the brain and liver of swine during prolonged severe HS. We conclude that there are multiple mechanisms by which pyruvate can possibly prevent cell damage caused during HS.

Previous studies have shown that the liver is the first organ to display signs of injury during hemorrhagic shock. We examined the mechanism by which pyruvate can prevent liver damage during hemorrhagic shock in swine anesthetized with halothane. Thirty minutes after the induction of a 240-min controlled arterial hemorrhage targeted at 40 mmHg, hypertonic sodium pyruvate (0.5 g · kg−1 · h−1) was infused to achieve an arterial concentration of 5 mM. The volume and osmolality effects of pyruvate were matched with 10% saline (HTS) and 0.9% saline (NS). Although the peak hemorrhage volume increased significantly in both the pyruvate and HTS group, only the pyruvate treatment was effective in delaying cardiovascular decompensation. In addition, pyruvate effectively maintained the NADH/NAD redox state, as evidenced by increased microdialysate pyruvate levels and a significantly lower lactate-to-pyruvate ratio. Pyruvate also prevented the loss of intracellular antioxidants (GSH) and a reduction in the GSH-to-GSSG ratio. These beneficial effects on the redox environment decreased hepatic cellular death by apoptosis. Pyruvate significantly increased the ratio of Bcl-Xl (antiapoptotic molecule)/Bax (proapoptotic molecule), prevented the release of cytochrome c from mitochondria, and decreased the fragmentation of caspase 3 and poly(ADP ribose) polymerase (DNA repair enzyme). These beneficial findings indicate that pyruvate infused 30 min after the onset of severe hemorrhagic shock is effective in maintaining the redox environment, preventing the loss of the key antioxidant GSH, and decreasing early apoptosis indicators.