Tag-free glycosylated rhMFG-E8 as a therapy for acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) can result from renal ischemia and reperfusion (I/R) and often occurs during surgical procedures in cardiac, liver, kidney transplantation and trauma-hemorrhage. Milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. Since MFG-E8 promotes clearance of apoptotic cells, we have explored its therapeutic potential in various organ injury conditions. To develop human MFG-E8 as a potential therapy, we have generated a human cell-expressed, and thus glycosylated, tag-free recombinant human (rh) MFG-E8 and tested its safety and biological activity in vitro. We hypothesize that the tag-free glycosylated rhMFG-E8 is protective in I/R-induced AKI and it can be developed as an effective therapy for AKI. METHODS: To assess the pharmacokinetic properties of the tag-free rhMFG-E8, Sprague Dawley rats were either untreated or treated with a bolus dose of the tag-free rhMFG-E8, blood collected at various time points and the recovery of human MFG-E8 in the blood were measured by ELISA. Adult male C57BL6 mice underwent bilateral renal ischemia for 30 min and immediately upon reperfusion, mice were treated intraperitoneally with either normal saline (vehicle) or 20 µg/kg human cell expressed, glycosylated tag-free rhMFG-E8. At either 24 h or 48 h after I/R, blood and kidneys were harvested for further analysis. In separate cohorts of mice after I/R and treatment, mice were observed for 10 days, and survival recorded. RESULTS: AKI rats treated with the tag-free rhMFG-E8 had similar half-life as those in the treated control rats. At 48 h after I/R-induced AKI, renal function markers, BUN and creatinine were increased and treatment with the tag-free rhMFG-E8 significantly decreased these markers. At both 24 h and 48 h after AKI, inflammatory cytokines, TNF-α, IL-6 and IL-1ß were increased and treatment decreased these levels. The kidney mRNA expressions of these cytokines were also increased at 24 h after AKI and treatment significantly decreased those mRNA expressions. Histologically, at 48 h after AKI, tubular damage, and the number of TUNEL staining cells were increased and treatment markedly decreased these measurements. Administration of tag-free rhMFG-E8 at the time of reperfusion improved survival in a 10-day survival study. CONCLUSION: Our new human cell-expressed tag-free rhMFG-E8 is protective in I/R-induced AKI and it may have the potential to be further developed as a safe and effective therapy for AKI.

Human cell-expressed tag-free rhMFG-E8 as an effective radiation mitigator.

Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and the possibility of antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 will have suitable structural and functional properties to be developed as a safe and effective novel biologic to treat inflammatory diseases including radiation injury. We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing an experimental rodent model of radiation injury, i.e., partial body irradiation (PBI). HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE with the standard human MFG-E8 loaded as control and, mass spectrometry followed by analysis using MASCOT for peptide mass fingerprint. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with a terminal elimination half-life in circulation of at least 1.45 h. In the 15 Gy PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 calculated using probit analysis was 1.058. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI suggesting it as a potential therapeutic candidate for a medical countermeasure for radiation injury. Our new human cell-expressed tag-free rhMFG-E8 has proper structural and functional properties to be further developed as a safe and effective therapy to treat victims of severe acute radiation injury.

A Synthetic Poly(A) Tail Targeting Extracellular CIRP Inhibits Sepsis.

Sepsis is an infectious inflammatory disease that often results in acute lung injury (ALI). Cold-inducible RNA-binding protein (CIRP) is an intracellular RNA chaperon that binds to mRNA's poly(A) tail. However, CIRP can be released in sepsis, and extracellular CIRP (eCIRP) is a damage-associated molecular pattern, exaggerating inflammation, ALI, and mortality. In this study, we developed an engineered poly(A) mRNA mimic, AAAAAAAAAAAA, named A12, with 2'-O-methyl ribose modification and terminal phosphorothioate linkages to protect it from RNase degradation, exhibiting an increased half-life. A12 selectively and strongly interacted with the RNA-binding motif of eCIRP, thereby preventing eCIRP's binding to its receptor, TLR4. In vitro treatment with A12 significantly decreased eCIRP-induced macrophage MAPK and NF-κB activation and inflammatory transcription factor upregulation. A12 also attenuated proinflammatory cytokine production induced by eCIRP in vitro and in vivo in macrophages and mice, respectively. We revealed that treating cecal ligation and puncture-induced sepsis with A12 significantly reduced serum organ injury markers and cytokine levels and ALI, and it decreased bacterial loads in the blood and peritoneal fluid, ultimately improving their survival. Thus, A12's ability to attenuate the clinical models of sepsis sheds lights on inflammatory disease pathophysiology and prevention of the disease progress.

Development of human cell-expressed tag-free rhMFG-E8 as a radiation mitigator and a therapeutic for acute kidney injury.

Background: Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and possible antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 can be developed as a safe and effective novel biologic to treat inflammatory diseases such as radiation injury and acute kidney injury (AKI). Methods: We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing two experimental rodent models of organ injury: partial body irradiation (PBI) and ischemia/reperfusion-induced AKI. Results: HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE analysis and mass spectrometry. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with an adequate half-life for therapeutic applications. In the PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 was 1.073. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI. In the model of AKI, tag-free rhMFG-E8 treatment significantly attenuated kidney injury and inflammation, and improved the 10-day survival. Conclusion: Our new human cell-expressed tag-free rhMFG-E8 can be further developed as a safe and effective therapy to treat victims of severe acute radiation injury and patients with acute kidney injury.

CBF oscillations induced by trigeminal nerve stimulation protect the pericontusional penumbra in traumatic brain injury complicated by hemorrhagic shock.

Traumatic peri-contusional penumbra represents crucial targets for therapeutic interventions after traumatic brain injury (TBI). Current resuscitative approaches may not adequately alleviate impaired cerebral microcirculation and, hence, compromise oxygen delivery to peri-contusional areas. Low-frequency oscillations in cerebral blood flow (CBF) may improve cerebral oxygenation in the setting of oxygen deprivation. However, no method has been reported to induce controllable oscillations in CBF and it hasn't been applied as a therapeutic strategy. Electrical stimulation of the trigeminal nerve (TNS) plays a pivotal role in modulating cerebrovascular tone and cerebral perfusion. We hypothesized that TNS can modulate CBF at the targeted frequency band via the trigemino-cerebrovascular network, and TNS-induced CBF oscillations would improve cerebral oxygenation in peri-contusional areas. In a rat model of TBI complicated by hemorrhagic shock, TNS-induced CBF oscillations conferred significant preservation of peri-contusional tissues leading to reduced lesion volume, attenuated hypoxic injury and neuroinflammation, increased eNOS expression, improved neurological recovery and better 10-day survival rate, despite not significantly increasing CBF as compared with those in immediate and delayed resuscitation animals. Our findings indicate that low-frequency CBF oscillations enhance cerebral oxygenation in peri-contusional areas, and play a more significant protective role than improvements in non-oscillatory cerebral perfusion or volume expansion alone.

Percutaneous Trigeminal Nerve Stimulation Induces Cerebral Vasodilation in a Dose-Dependent Manner.

BACKGROUND: The trigeminal nerve directly innervates key vascular structures both centrally and peripherally. Centrally, it is known to innervate the brainstem and cavernous sinus, whereas peripherally the trigemino-cerebrovascular network innervates the majority of the cerebral vasculature. Upon stimulation, it permits direct modulation of cerebral blood flow (CBF), making the trigeminal nerve a promising target for the management of cerebral vasospasm. However, trigeminally mediated cerebral vasodilation has not been applied to the treatment of vasospasm. OBJECTIVE: To determine the effect of percutaneous electrical stimulation of the infraorbital branch of the trigeminal nerve (pTNS) on the cerebral vasculature. METHODS: In order to determine the stimulus-response function of pTNS on cerebral vasodilation, CBF, arterial blood pressure, cerebrovascular resistance, intracranial pressure, cerebral perfusion pressure, cerebrospinal fluid calcitonin gene-related peptide (CGRP) concentrations, and the diameter of cerebral vessels were measured in healthy and subarachnoid hemorrhage (SAH) rats. RESULTS: The present study demonstrates, for the first time, that pTNS increases brain CGRP concentrations in a dose-dependent manner, thereby producing controllable cerebral vasodilation. This vasodilatory response appears to be independent of the pressor response induced by pTNS, as it is maintained even after transection of the spinal cord at the C5-C6 level and shown to be confined to the infraorbital nerve by administration of lidocaine or destroying it. Furthermore, such pTNS-induced vasodilatory response of cerebral vessels is retained after SAH-induced vasospasm. CONCLUSION: Our study demonstrates that pTNS is a promising vasodilator and increases CBF, cerebral perfusion, and CGRP concentration both in normal and vasoconstrictive conditions.

Recombinant human milk fat globule-EGF factor VIII (rhMFG-E8) as a therapy for sepsis after acute exposure to alcohol.

BACKGROUND: Alcohol intake predisposes to infections and sepsis. Alcohol and sepsis inhibit the expression of milk fat globule epidermal growth factor-factor VIII (MFG-E8), a glycoprotein essential for optimal efferocytosis, resulting in the release of proinflammatory molecules and increased sepsis severity. We previously reported that recombinant mouse (rm) MFG-E8 attenuates sepsis-induced organ injury in rats with acute alcohol intoxication. In order to develop a therapy that can be safely used in humans, we have produced recombinant human (rh) MFG-E8 and evaluated its efficacy to ameliorate sepsis after acute exposure to alcohol. METHODS: We induced acute alcohol intoxication with a bolus injection of alcohol (1.75 g/kg BW) followed by an intravenous infusion of 300 mg/kg/h alcohol for 10 h. Sepsis was then induced by cecal ligation and puncture (CLP). At -10, 0, and 10 h relative to CLP, rats received MFG-E8 or vehicle (albumin) intravenously. Animals were euthanized at 20 h after CLP for blood and tissue collection. Additional groups of animals were used for a survival study. RESULTS: Compared to vehicle, rhMFG-E8 treatment ameliorated blood levels of proinflammatory cytokines (% improvement: TNF-α 49.8%, IL-6 34.7%) and endotoxin (61.7%), as well as of transaminases (AST 36.2%, ALT 40.1%) and lactate (18.4%). Rats treated with rhMFG-E8 also had a significant histological attenuation of the acute lung injury, as well as a reduction in the number of apoptotic cells in the thymus (43.4%) and cleaved caspase 3 (38.7%) in the spleen. In addition, rhMFG-E8 improved the 10-day sepsis survival rate from 45 to 80% CONCLUSION: rhMFG-E8 significantly ameliorated sepsis in rats with acute alcohol exposure, demonstrating rhMFG-E8's potential to be developed as an effective therapy for sepsis in alcohol abusers.

Trigeminal Nerve Stimulation: A Novel Method of Resuscitation for Hemorrhagic Shock.

OBJECTIVES: To determine if trigeminal nerve stimulation can ameliorate the consequences of acute blood loss and improve survival after severe hemorrhagic shock. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Severe hemorrhagic shock was induced in rats by withdrawing blood until the mean arterial blood pressure reached 27 ± 1 mm Hg for the first 5 minutes and then maintained at 27 ± 2 mm Hg for 30 minutes. The rats were randomly assigned to either control, vehicle, or trigeminal nerve stimulation treatment groups. The effects of trigeminal nerve stimulation on survival rate, autonomic nervous system activity, hemodynamics, brain perfusion, catecholamine release, and systemic inflammation after severe hemorrhagic shock in the absence of fluid resuscitation were analyzed. MEASUREMENTS AND MAIN RESULTS: Trigeminal nerve stimulation significantly increased the short-term survival of rats following severe hemorrhagic shock in the absence of fluid resuscitation. The survival rate at 60 minutes was 90% in trigeminal nerve stimulation treatment group whereas 0% in control group (p < 0.001). Trigeminal nerve stimulation elicited strong synergistic coactivation of the sympathetic and parasympathetic nervous system as measured by heart rate variability. Without volume expansion with fluid resuscitation, trigeminal nerve stimulation significantly attenuated sympathetic hyperactivity paralleled by increase in parasympathetic tone, delayed hemodynamic decompensation, and improved brain perfusion following severe hemorrhagic shock. Furthermore, trigeminal nerve stimulation generated sympathetically mediated low-frequency oscillatory patterns of systemic blood pressure associated with an increased tolerance to central hypovolemia and increased levels of circulating norepinephrine levels. Trigeminal nerve stimulation also decreased systemic inflammation compared with the vehicle. CONCLUSIONS: Trigeminal nerve stimulation was explored as a novel resuscitation strategy in an animal model of hemorrhagic shock. The results of this study showed that the stimulation of trigeminal nerve modulates both sympathetic and parasympathetic nervous system activity to activate an endogenous pressor response, improve cerebral perfusion, and decrease inflammation, thereby improving survival.

Neuroprotective Effects of Trigeminal Nerve Stimulation in Severe Traumatic Brain Injury.

Following traumatic brain injury (TBI), ischemia and hypoxia play a major role in further worsening of the damage, a process referred to as 'secondary injury'. Protecting neurons from causative factors of secondary injury has been the guiding principle of modern TBI management. Stimulation of trigeminal nerve induces pressor response and improves cerebral blood flow (CBF) by activating the rostral ventrolateral medulla. Moreover, it causes cerebrovasodilation through the trigemino-cerebrovascular system and trigemino-parasympathetic reflex. These effects are capable of increasing cerebral perfusion, making trigeminal nerve stimulation (TNS) a promising strategy for TBI management. Here, we investigated the use of electrical TNS for improving CBF and brain oxygen tension (PbrO2), with the goal of decreasing secondary injury. Severe TBI was produced using controlled cortical impact (CCI) in a rat model, and TNS treatment was delivered for the first hour after CCI. In comparison to TBI group, TBI animals with TNS treatment demonstrated significantly increased systemic blood pressure, CBF and PbrO2 at the hyperacute phase of TBI. Furthermore, rats in TNS-treatment group showed significantly reduced brain edema, blood-brain barrier disruption, lesion volume, and brain cortical levels of TNF-α and IL-6. These data provide strong early evidence that TNS could be an effective neuroprotective strategy.

A New Approach for On-Demand Generation of Various Oxygen Tensions for In Vitro Hypoxia Models.

The development of in vitro disease models closely mimicking the functions of human disease has captured increasing attention in recent years. Oxygen tensions and gradients play essential roles in modulating biological systems in both physiologic and pathologic events. Thus, controlling oxygen tension is critical for mimicking physiologically relevant in vivo environments for cell, tissue and organ research. We present a new approach for on-demand generation of various oxygen tensions for in vitro hypoxia models. Proof-of-concept prototypes have been developed for conventional cell culture microplate by immobilizing a novel oxygen-consuming biomaterial on the 3D-printed insert. For the first time, rapid (~3.8 minutes to reach 0.5% O2 from 20.9% O2) and precisely controlled oxygen tensions/gradients (2.68 mmHg per 50 µm distance) were generated by exposing the biocompatible biomaterial to the different depth of cell culture media. In addition, changing the position of 3D-printed inserts with immobilized biomaterials relative to the cultured cells resulted in controllable and rapid changes in oxygen tensions (<130 seconds). Compared to the current technologies, our approach allows enhanced spatiotemporal resolution and accuracy of the oxygen tensions. Additionally, it does not interfere with the testing environment while maintaining ease of use. The elegance of oxygen tension manipulation introduced by our new approach will drastically improve control and lower the technological barrier of entry for hypoxia studies. Since the biomaterials can be immobilized in any devices, including microfluidic devices and 3D-printed tissues or organs, it will serve as the basis for a new generation of experimental models previously impossible or very difficult to implement.

MFG-E8 inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG­E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG­E8 attenuates neutrophil migration. Recombinant human MFG­E8 (rhMFG­E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL­60, was treated with rhMFG­E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin­8 (IL­8) as the chemoattractant. Surface CXCR2 and intracellular G protein­coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen­activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG­E8 resulted in a significant inhibition of dHL­60 cell migration in a dose­dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG­E8­treated dHL­60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL­60 cells, treatment with rhMFG­E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10­30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL­60 cell migration which was significantly inhibited treatment with rhMFG­E8. Furthermore, blocking the MFG­E8 receptors, αvß3/αvß5­integrins, by anti­αv­integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG­E8­induced inhibition of dHL­60 cell migration. Finally, treatment of the dHL­60 cells with SB203580 and PD98059 neutralized the rhMFG­E8­induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG­E8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

Cold-inducible RNA-binding protein is an important mediator of alcohol-induced brain inflammation.

Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP(-/-)) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP(-/-) mice. Brain TNF-α mRNA and protein expressions along with IL-1ß protein levels were significantly increased in WT mice, which was not seen in the CIRP(-/-) mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1ß were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.

Mitochondrial transcription factor A is a proinflammatory mediator in hemorrhagic shock.

Endogenous molecules released by dying cells [i.e., damage-associated molecular patterns (DAMPs)] after trauma and severe blood loss can activate pattern recognition receptors, leading to a cascade of inflammatory responses and organ injury. Mitochondrial transcription factor A (TFAM) is a transcription factor for mitochondrial DNA. TFAM is structurally related to high mobility group box 1 (HMGB1), an important member of DAMPs. We, therefore, hypothesized that TFAM can be released into the circulation after hemorrhage to initiate inflammatory responses. In order to examine this hypothesis, male Sprague-Dawley rats were bled to and maintained at a mean arterial pressure of 40 mmHg for 90 min. They were then resuscitated with an equal volume of shed blood in the form of Ringer's lactate (i.e., low-volume resuscitation) over 60 min. TFAM levels in the serum were measured at 4 h after hemorrhage and resuscitation. Our results showed that serum levels of TFAM were more than doubled after hemorrhage and resuscitation. To further characterize TFAM's biological activity, we expressed recombinant rat TFAM with a GST-tag (GST-TFAM) in an E. coli expression system. The purity of GST-TFAM was over 99% and it was immunoreactive for specific anti-TFAM antibodies. Using RAW 264.7 cells and primary rat peritoneal macrophages, we showed that GST-TFAM dose-dependently increased TNF-α release. To determine the biological activity of GST-TFAM in vivo, GST-TFAM was intravenously injected in healthy male adult rats. Our results demonstrated that intravenous injection of GST-TFAM, not GST alone, upregulated circulating levels of pro-inflammatory cytokines, increased neutrophil infiltration to the lungs and caused organ injury in healthy animals. Thus, TFAM can act as a DAMP and may contribute to the initiation of inflammatory responses in hemorrhagic shock.

Ghrelin maintains the cardiovascular stability in severe sepsis.

BACKGROUND: Cardiovascular dysfunction, characterized by reduced cardiac contractility and depressed endothelium-dependent vascular relaxation, is common in severe sepsis. Although it is known that ghrelin produces beneficial effects following various adverse circulatory conditions, it remains unknown whether ghrelin increases cardiac contractility and improves vascular responsiveness to vasoactive agents in severe sepsis. METHODS: Male adult rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed mini-pump over 15 h. At 20 h after CLP (i.e., severe sepsis), the maximal rates of ventricular pressure increase (+dP/dt(max)) and decrease (-dP/dt(max)) were determined in vivo. In additional groups of animals, the thoracic aortae were isolated at 20 h after CLP. The aortae were cut into rings, and placed in organ chambers. Norepinephrine (NE) was used to induce vascular contraction. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerine (NTG) were carried out. RESULTS: +dP/dt(max) and -dP/dt(max) decreased significantly at 20 h after CLP. Treatment with ghrelin significantly increased +dP/dt(max) and -dP/dt(max) by 36% (P < 0.05) and 35% (P < 0.05), respectively. Moreover, NE-induced vascular contraction and endothelium-dependent (ACh-induced) vascular relaxation decreased significantly at 20 h after CLP. Administration of ghrelin, however, increased NE-induced vascular contraction and ACh-induced vascular relaxation. In contrast, no significant reduction in NTG-induced vascular relaxation was seen in rats with severe sepsis irrespective of ghrelin treatment. CONCLUSIONS: Ghrelin may be further developed as a useful agent for maintaining cardiovascular stability in severe sepsis.

Recombinant human MFG-E8 attenuates cerebral ischemic injury: its role in anti-inflammation and anti-apoptosis.

Excessive inflammation and apoptosis contribute to the pathogenesis of ischemic stroke. MFG-E8 is a 66-kDa glycoprotein that has shown tissue protection in various models of organ injury. However, the potential role of MFG-E8 in cerebral ischemia has not been investigated. We found that levels of MFG-E8 protein in the brain were reduced at 24 h after cerebral ischemia. To assess the potential role of MFG-E8 in cerebral ischemia, adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO). At 1 h post-stroke onset, an intravenous administration of 1 ml saline as vehicle or 160 µg/kg BW recombinant human MFG-E8 (rhMFG-E8) as treatment was given. The optimal dose of rhMFG-E8 was obtained from previous dose-response organ protection in rat sepsis studies. Neurological scores were determined at 24 h and 48 h post-MCAO. Rats were sacrificed thereafter and brains rapidly removed and analyzed for infarct size, histopathology, and markers of inflammation and apoptosis. Compared with saline vehicle, rhMFG-E8 treatment led to significant decreases in sensorimotor and vestibulomotor deficits, and infarct size at 24 h and 48 h post-MCAO. Measures associated with improved outcome included reduced microglial inflammatory cytokine secretion, adhesion molecules and neutrophil influx, cleaved caspase-3, and upregulation of peroxisome proliferator activated receptor-γ (PPAR-γ), and Bcl-2/Bax ratio leading to decreased apoptosis. Thus, rhMFG-E8 treatment is neuroprotective against cerebral ischemia through suppression of inflammation and apoptosis. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Expression and characterization of recombinant human milk fat globule-EGF factor VIII.

Apoptosis plays an important role in the patho-biology of sepsis. The opsonizing protein milk fat globule-EGF factor VIII (MFG-E8) is involved in apoptotic cell clearance. Our previous studies have shown that administration of rat MFG-E8-containing exosomes or recombinant murine MFG-E8 (rmMFG-E8) is protective in a rat model of sepsis induced by cecal ligation of puncture (CLP). However, one obstacle hampering the development of MFG-E8 as a therapeutic agent for septic patients is the potential immunogenicity of animal proteins in humans. The purpose of this study, therefore, was to express recombinant human MFG-E8 (rhMFG-E8) and characterize its biological activity. Using an E. coli system, we successfully expressed and purified the mature molecule of human MFG-E8 (Leu24-Cys387). The purity of rhMFG-E8 was over 99% and it was immunoreactive for specific anti-human MFG-E8 antibodies. Amino acid sequence analysis by LC-MS/MS identified the purified protein as human MFG-E8. Using primary rat peritoneal macrophages, we showed that rhMFG-E8 markedly increased peritoneal macrophage phagocytosis of apoptotic thymocytes, which was as effective as commercial rmMFG-E8. To determine the biological activity of rhMFG-E8 in vivo, male adult rats were subjected to sepsis by CLP. rhMFG-E8 or rmMFG-E8 were administered intravenously at the time of CLP. Our results demonstrated that both rhMFG-E8 and rmMFG-E8 reduced thymocyte apoptosis and plasma levels of lactate and IL-6 at 20 h after CLP, and improved the 10-day survival rate. Thus, we have successfully expressed and purified biologically active rhMFG-E8. Our newly-expressed rhMFG-E8 is highly effective in the rat model of sepsis.

Peripheral administration of human adrenomedullin and its binding protein attenuates stroke-induced apoptosis and brain injury in rats.

Stroke is a leading cause of death and the primary medical cause of acquired adult disability worldwide. The progressive brain injury after acute stroke is partly mediated by ischemia-elicited inflammatory responses. The vasoactive hormone adrenomedullin (AM), upregulated under various inflammatory conditions, counterbalances inflammatory responses. However, regulation of AM activity in ischemic stroke remains largely unknown. Recent studies have demonstrated the presence of a specific AM binding protein (that is, AMBP-1) in mammalian blood. AMBP-1 potentiates AM biological activities. Using a rat model of focal cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO), we found that plasma levels of AM increased significantly, whereas plasma levels of AMBP-1 decreased significantly after stroke. When given peripherally early after MCAO, exogenous human AM in combination with human AMBP-1 reduced brain infarct volume 24 and 72 h after MCAO, an effect not observed after the treatment by human AM or human AMBP-1 alone. Furthermore, treatment of human AM/AMBP-1 reduced neuron apoptosis and morphological damage, inhibited neutrophil infiltration in the brain and decreased serum levels of S100B and lactate. Thus, human AM/AMBP-1 has the ability to reduce stroke-induced brain injury in rats. AM/AMBP-1 can be developed as a novel therapeutic agent for patients with ischemic stroke.

Upregulation of Kupffer cell α2A-Adrenoceptors and downregulation of MKP-1 mediate hepatic injury in chronic alcohol exposure.

Alcohol-induced liver disease is associated with unacceptable morbidity and mortality. When activated, Kupffer cells (KCs), the resident macrophages in the liver, release proinflammatory cytokine TNF-α, a key mediator of hepatic damage. Although chronic alcohol causes increase in norepinephrine (NE) release leading to hepatic dysfunction, the mechanism of NE-induced hepatic injury in chronic alcohol exposure has not been elucidated. This study was conducted to determine whether chronic alcohol exposure increases NE and upregulates KC α(2A)-adrenoceptors (α(2A)-AR) to cause TNF-α release. We also examined the role of mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1) in this process. Male adult rats were fed the Lieber-DeCarli liquid diet containing alcohol as 36% of total calories. The animals were sacrificed after 6 weeks and blood and liver samples were harvested for further analysis. KCs from healthy male rats were cultured with alcohol for 7 days, and cells then harvested for RNA and protein analyses. Chronic alcohol exposure resulted in hepatic damage. Alcohol caused a 276% increase in circulating NE and 86% increase in TNF-α in the liver. There was a 75% and 62% decrease in MKP-1 mRNA and protein levels, respectively in the liver. In-vitro experiments revealed 121% and 98% increase in TNF-α and α(2A)-AR mRNA levels with alcohol exposure, respectively, and a 32% decrease in MKP-1 mRNA compared to controls. In summary, chronic alcohol exposure elevates NE and upregulates KC α(2A)-AR to release TNF-α. Alcohol induced downregulation of MKP-1 leads to further release of TNF-α and hepatic injury.

Milk fat globule EGF factor 8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.

BACKGROUND: Despite advances in our understanding of excessive alcohol-intake-related tissue injury and modernization of the management of septic patients, high morbidity and mortality caused by infectious diseases in alcohol abusers remain a prominent challenge. Our previous studies have shown that milk fat globule epidermal growth factor-factor VIII (MFG-E8), a protein required to opsonize apoptotic cells for phagocytosis, is protective in inflammation. However, it remains unknown whether MFG-E8 ameliorates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats. The purpose of this study was to determine whether recombinant murine MFG-E8 (rmMFG-E8) attenuates organ injury after acute alcohol exposure and subsequent sepsis. METHODS: Acute alcohol intoxication was induced in male adult rats by a bolus injection of intravenous alcohol at 1.75 g/kg BW, followed by an intravenous infusion of 300 mg/kg BW/h of alcohol for 10 hours. Sepsis was induced at the end of 10-hour alcohol infusion by cecal ligation and puncture (CLP). rmMFG-E8 or vehicle (normal saline) was administered intravenously 3 times (i.e., at the beginning of alcohol injection, the beginning of CLP, and 10 hours post-CLP) at a dose of 20 microg/kg BW each. Blood and tissue samples were collected 20 hours after CLP in alcoholic animals for various measurements. RESULTS: Acute alcohol exposure per se did not affect the production of MFG-E8; however, it primed the animal and enhanced sepsis-induced MFG-E8 downregulation in the spleen. Administration of rmMFG-E8 reduces alcohol/sepsis-induced apoptosis in the spleen, lungs, and liver. In addition, administration of rmMFG-E8 after alcohol exposure and subsequent sepsis decreases circulating levels of TNF-alpha and interleukin-6 and attenuates organ injury. CONCLUSIONS: rmMFG-E8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.

Milk fat globule epidermal growth factor 8 attenuates acute lung injury in mice after intestinal ischemia and reperfusion.

RATIONALE: Milk fat globule epidermal growth factor 8 (MFG-E8) is a potent opsonin for the clearance of apoptotic cells and is produced by mononuclear cells of immune competent organs including the spleen and lungs. It attenuates chronic and acute inflammation such as autoimmune glomerulonephritis and bacterial sepsis by enhancing apoptotic cell clearance. Ischemia-reperfusion (I/R) injury of the gut results in severe inflammation, apoptosis, and remote organ damage, including acute lung injury (ALI). OBJECTIVES: To determine whether MFG-E8 attenuates intestinal and pulmonary inflammation after gut I/R. METHODS: Wild-type (WT) and MFG-E8(-/-) mice underwent superior mesenteric artery occlusion for 90 minutes, followed by reperfusion for 4 hours. A group of WT mice was treated with 0.4 microg/20 g recombinant murine MFG-E8 (rmMFG-E8) at the beginning of reperfusion. Four hours after reperfusion, MFG-E8, cytokines, myeloperoxidase activity, apoptosis, and histopathology were assessed. A 24-hour survival study was conducted in rmMFG-E8- and vehicle-treated WT mice. MEASUREMENTS AND MAIN RESULTS: Mesenteric I/R caused severe widespread injury and inflammation of the small intestines and remote organs, including the lungs. MFG-E8 levels decreased in the spleen and lungs by 50 to 60%, suggesting impaired apoptotic cell clearance. Treatment with rmMFG-E8 significantly suppressed inflammation (TNF-alpha, IL-6, IL-1beta, and myeloperoxidase) and injury of the lungs, liver, and kidneys. MFG-E8-deficient mice suffered from greatly increased inflammation and potentiated ALI, whereas treatment with rmMFG-E8 significantly improved the survival in WT mice. CONCLUSIONS: MFG-E8 attenuates inflammation and ALI after gut I/R and may represent a novel therapeutic agent.