Adrenomedullin binding protein-1 modulates vascular responsiveness to adrenomedullin in late sepsis

Adrenomedullin (AM), a potent vasodilatory peptide, plays an important role in initiating the hyperdynamic response during the early stage of sepsis. Moreover, the reduced vascular responsiveness to AM appears to be responsible for the transition from the early, hyperdynamic to the late, hypodynamic phase of sepsis. Although the novel specific AM binding protein-1 (AMBP-1) enhances AM-mediated action in a cultured cell line, it remains to be determined whether AMBP-1 plays any role in modulating vascular responsiveness to AM during sepsis. To study this, adult male rats were subjected to sepsis by cecal ligation and puncture (CLP). The thoracic aorta was harvested for determination of AM-induced vascular relaxation. Aortic levels of AMBP-1 were determined by Western blot analysis, and AM receptor gene expression in the aortic tissue was assessed by RT-PCR. The results indicate that AMBP-1 significantly enhanced AM-induced vascular relaxation in aortic rings from sham-operated animals. Although vascular responsiveness to AM decreased at 20 h after CLP (i.e., the late, hypodynamic stage of sepsis), addition of AMBP-1 in vitro restored the vascular relaxation induced by AM. Moreover, the aortic level of AMBP-1 decreased significantly at 20 h after CLP. In contrast, AM receptor gene expression was not altered under such conditions. These results, taken together, suggest that AMBP-1 plays an important role in modulating vascular responsiveness to AM, and the reduced AMBP-1 appears to be responsible for the vascular AM hyporesponsiveness observed during the hypodynamic phase of sepsis.     

Andrenomedullin and cardiovascular responses in sepsis.

The typical cardiovascular response to polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Although the factors and/or mediators responsible for producing the transition from the hyperdynamic to the hypodynamic stage are not fully understood, recent studies have suggested that adrenomedullin (AM), a potent vasodilatory peptide, appears to play an important role in initiating the hyperdynamic response following the onset of sepsis. In addition, the reduced vascular responsiveness to AM may result in the transition from the early, hyperdynamic phase to the late, hypodynamic phase of sepsis. It is possible that changes in newly reported AM receptors calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-2 or -3 (RAMP2, RAMP3) as well as AM binding protein-1 (AMBP-1) may also play distinct roles in the biphasic cardiovascular response observed during sepsis. Although it remains unknown whether AM gene delivery or a chronic increase in vascular AM production in transgenic animals attenuates the development of hypodynamic sepsis and septic shock, it has been shown that modulation of AM vascular responsiveness with pharmacologic agents reduces sepsis-induced mortality. It has been recently demonstrated that AMBP-1 enhances AM's physiologic effects and plasma levels of AMBP-1 decrease following infections. We therefore propose that downregulation of AMBP-1 and the reduced AM receptor responsiveness are crucial factors responsible for the transition from the hyperdynamic phase to the hypodynamic phase of sepsis.     

Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-alpha from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-alpha release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-alpha production from RAW cells by 38-63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-alpha release by 14-22% and 13-22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-alpha production (by 24-35%). Moreover, the upregulated TNF-alpha mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-alpha production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-alpha production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-alpha mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-alpha expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-alpha may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.     

Vasoactive hormone adrenomedullin and its binding protein: anti-inflammatory effects by up-regulating peroxisome proliferator-activated receptor-gamma

Sepsis is a critical inflammatory condition from which numerous patients die due to multiple organ failure and septic shock. The vasoactive hormone adrenomedullin (AM) and its binding protein (AMBP-1) are beneficial in sepsis by abrogating the progression to irreversible shock and decreasing proinflammatory cytokine release. To investigate the anti-inflammatory mechanism, we studied to determine the effect of the AM/AMBP-1 complex on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-gamma expression in vivo and in vitro and was associated with increased TNF-alpha production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-gamma levels in both models, resulting in TNF-alpha suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-alpha production in the absence of PPAR-gamma. LPS caused the suppression of intracellular cyclic AMP (cAMP), which was prevented by simultaneous AM/AMBP-1 treatment. Although incubation with dibutyryl cAMP significantly decreased LPS-induced TauNuF-alpha release, it did not alter PPAR-gamma expression. Through inhibition studies using genistein and PD98059 we found that the Pyk-2 tyrosine kinase-ERK1/2 pathway is in part responsible for the AM/AMBP-1-mediated induction of PPAR-gamma and the anti-inflammatory effect. We conclude that AM/AMBP-1 is protective in sepsis due to its vasoactive properties and direct anti-inflammatory effects mediated through both the cAMP-dependent pathway and Pyk-2-ERK1/2-dependent induction of PPAR-gamma.     

Circulating hormone adrenomedullin and its binding protein protect neural cells from hypoxia-induced apoptosis

Background

Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. Neural cells exposed to ischemia can undergo apoptosis. Adrenomedullin (AM) in combination with its enhancing binding protein, AMBP-1, has been shown to reduce tissue damage in inflammation.

Methods

To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells.

Results

After exposure to 1% O₂ for 20 h, neural cells were injured with decreased ATP levels and increased LDH release. Pre-administration of AM/AMBP-1 significantly reduced hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3, compared to cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, an elevation of cAMP levels by forskolin protected neural cells from hypoxia-induced injury. Inhibition of PKA by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis.

Conclusions

AM/AMBP-1 elevates cAMP levels, followed by activating PKA, to protect neural cells from the injury caused by hypoxia.

General significance

AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.     

Purification and characterization of human adrenomedullin binding protein-1

We recently discovered that the vascular responsiveness to adrenomedullin (AM), a potent vasoactive peptide, decreased during sepsis and hemorrhage in the rat and was markedly improved by its novel binding protein (AMBP-1). Moreover, AM/AMBP-1 appears to be one of the leading candidates for further development to treat sepsis and hemorrhage. However, the extremely high cost of commercial AMBP-1 limits the development of human AM and AMBP-1 as therapeutic agents. The purpose of this study was to isolate and purify AMBP-1 from normal human serum and test its stability and biological activity under in vitro and in vivo conditions. AMBP-1 was isolated and purified from normal human serum with a yield of about 3.0 mg per 100 mL and purity of >99%. The purified AMBP-1 has a AM-binding capacity similar to that of the commercial AMBP-1. Human AM and human AMBP-1 in combination significantly inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production from macrophages. The biological activity of the purified human AMBP-1 was well preserved when stored at 45 degrees C for 5 d in solution or at 100 degrees C for 1 h in powder. Moreover, administration of AM and purified AMBP-1 to hemorrhaged rats attenuated tissue injury and neutrophil accumulation. Purified AMBP-1 in combination with AM also suppressed the hemorrhage-induced rise in serum cytokines TNF-alpha and IL-6. Thus, we have successfully purified biologically active AMBP-1 from human normal serum and demonstrated the stability of purified human AMBP-1. This technique will enable us to further develop human AM/AMBP-1 as a novel treatment for safe and effective therapy of patients with hemorrhagic shock, sepsis, and ischemic injury.     

Adrenomedullin and adrenomedullin binding protein-1 protect endothelium-dependent vascular relaxation in sepsis

Downregulation of vascular endothelial constitutive nitric oxide synthase (ecNOS) contributes to the vascular hyporesponsiveness in sepsis. Although coadministration of the potent vasodilatory peptide adrenomedulin (AM) and the newly discovered AM binding protein (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis, it remains unknown whether AM/AMBP-1 prevents endothelial cell dysfunction. To investigate this possibility, we subjected adult male rats to sepsis by cecal ligation and puncture (CLP), with or without subsequent intravenous administration of the combination of AM (12 microg/kg) and AMBP-1 (40 microg/kg). Thoracic aortae were harvested 20 h after CLP (i.e., the late stage of sepsis) and endothelium-dependent vascular relaxation was determined by the addition of acetylcholine (ACh) in an organ bath system. In addition, ecNOS gene and protein expression was assessed by RT-PCR and immunohistochemistry, respectively. The results indicate that ACh-induced (i.e., endothelium-dependent) vascular relaxation was significantly reduced 20 h after CLP. Administration of AM/AMBP-1 prevented the reduction of vascular relaxation. In addition, ecNOS gene expression in aortic and pulmonary tissues was downregulated 20 h after CLP and AM/AMBP-1 attenuated such a reduction. Moreover, the decreased ecNOS staining in thoracic aortae of septic animals was prevented by the treatment with AM/AMBP-1. These results, taken together, indicate that AM/AMBP-1 preserves ecNOS and prevents reduced endothelium-dependent vascular relaxation (i.e., endothelial cell dysfunction) in sepsis. In light of our recent finding that AM/AMBP-1 improves organ function and reduces mortality in sepsis, it is most likely that the protective effect of these compounds on ecNOS is a mechanism responsible for the salutary effect of AM/AMBP-1 in sepsis.     

Kupffer cell products and interleukin 1beta directly promote VLDL secretion and apoB mRNA up-regulation in rodent hepatocytes

Plasma VLDL accumulation in Gram-negative sepsis is partly ascribed to an increased hepatic VLDL production driven by pro-inflammatory cytokines. We previously showed that hepatocytes of the Kupffer cell (KC)-rich periportal area are major contributors to enhanced VLDL production in lipopolysaccharide (LPS)-injected rats. However, it remains to be established whether KC generated products directly affect the number (apoB) and composition of secreted VLDL. Using rat primary cells, we show here that hepatocytes respond to stimulation by soluble mediators released by LPS-stimulated Kupffer cells with enhanced secretion of apoB and triglycerides in phospholipid-rich VLDL particles. Unstimulated KC products also augmented the secretion of normal VLDL, doubling apoB mRNA abundance. IL-1beta treatment resulted in concentration-dependent increases of hepatocyte apoB mRNA and protein secretion, increases that were greater, but not additive, when combined with IL-6 and TNF-alpha. Lipid secretion and MTP mRNA levels were unaffected by cytokines. In summary: (i) enhanced secretion of phospholipid-rich VLDL particles is a net hepatocyte response to LPS-stimulated KC products, which gives a clue about the local role of Kupffer cells in septic dyslipidemia induction; and (ii) pro-inflammatory cytokines act redundantly to enhance apoB secretion involving translational apoB up-regulation, but other humoral components or KC mediators are necessary to accomplish increased lipid association.     

Human adrenomedullin combined with human adrenomedullin binding protein-1 is protective in gut ischemia and reperfusion injury in the rat

Previous studies have demonstrated that co-administration of rat adrenomedullin (AM) and human AM binding protein-1 (AMBP-1) has various beneficial effects following adverse circulatory conditions. In order to reduce rat proteins to elicit possible immune responses in humans, we determined the effect of human AM combined with human AMBP-1 after intestinal ischemia and reperfusion (I/R). Intestinal ischemia was induced in the rat by occluding the superior mesenteric artery for 90 min. At 60 min after the beginning of reperfusion, human AM/AMBP-1 at 3 different dosages was administered intravenously over 30 min. At 240 min after the treatment, blood and tissue samples were harvested and measured for pro-inflammatory cytokines (i.e., TNF-alpha and IL-6), myeloperoxidase activities in the gut and lungs, and cleaved caspase-3 expression in the lungs, as well as serum levels of hepatic enzymes and lactate. In additional groups of animals, a 10-day survival study was conducted. Results showed that administration of human AM/AMBP-1 reduced pro-inflammatory cytokines, attenuated organ injury, and improved the survival rate in a seemingly dose-response fashion. Co-administration of the highest dose of human AM/AMBP-1 in this study had the optimal therapeutic effect in the rat model of intestinal I/R.     

Oxidative products from alcohol metabolism differentially modulate pro-inflammatory cytokine expression in Kupffer cells and hepatocytes

Pro-inflammatory cytokines play a vital role in the pathogenesis of alcoholic steatohepatitis. The present study was to determine the role of alcohol-induced oxidative stress in modulating cytokine production. A rat model of alcohol consumption was used to determine alcohol-induced hepatic cytokine expression. Chronic alcohol exposure caused lipid accumulation, oxidative stress, and inflammation in the livers of Wistar rats. The role of oxidative stress in regulating cell type-specific cytokine production was further dissected in vitro. Lipopolysaccharide (LPS) dose-dependently upregulated TNF-α, MIP-1α, MCP-1, and CINC-1 in Kupffer cells-SV40, whereas TNF-α dose-dependently induced CINC-1, IP-10, and MIP-2 expression in H4IIEC3 hepatoma cells. An additive effect on cytokine production was observed in both Kupffer cells-SV40 and hepatocytes when combined hydrogen peroxide with LPS or TNF-α, respectively, which was associated with NF-κB activation and histone H3 hyper-acetylation. Unexpectedly, an inhibitory effect of 4-hydroxynonenal on cytokine production was revealed in LPS-treated Kupffer cells-SV40. Mechanistic study showed that 4-hydroxynonenal significantly enhanced mRNA degradation of TNF-α, MCP-1, and MIP-1α, and decreased the protein levels of MCP-1 in LPS-stimulated Kupffer cells-SV40 through reducing the phosphorylation of mRNA binding proteins. This study suggests that Kupffer cells and hepatocytes express distinct pro-inflammatory cytokines/chemokines in response to alcohol intoxication, and oxidative products (4-hydroxynonenal) differentially modulate pro-inflammatory cytokine/chemokine production via NF-κB signaling, histone acetylation, and mRNA stability.     

Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock

We recently discovered that vascular responsiveness to adrenomedullin (AM), a vasoactive hormone, decreases after hemorrhage, which is markedly improved by the addition of its binding protein AMBP-1. One obstacle hampering the development of AM/AMBP-1 as resuscitation agents in trauma victims is the potential immunogenicity of rat proteins in humans. Although less potent than rat AM, human AM has been shown to increase organ perfusion in rats. We therefore hypothesized that administration of human AM/AMBP-1 improves organ function and survival after severe blood loss in rats. To test this, male Sprague-Dawley rats were bled to and maintained at an MAP 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. At 15 min after the beginning of resuscitation, human AM/AMBP-1 (12/40 or 48/160 microg/kg BW) were administered intravenously over 45 min. Various pathophysiological parameters were measured 4h after resuscitation. In additional groups of animals, a 12-day survival study was conducted. Our result showed that tissue injury as evidenced by increased levels of transaminases, lactate, and creatinine, was present at 4h after hemorrhage and resuscitation. Moreover, pro-inflammatory cytokines TNF-alpha and IL-6 were also significantly elevated. Administration of AM/AMBP-1 markedly attenuated tissue injury, reduced cytokine levels, and improved the survival rate from 29% (vehicle) to 62% (low-dose) or 70% (high-dose). However, neither human AM alone nor human AMBP-1 alone prevented the significant increase in ALT, AST, lactate and creatinine at 4h after the completion of hemorrhage and resuscitation. Moreover, the half-life of human AM and human AMBP-1 in rats was 35.8 min and 1.68 h, respectively. Thus, administration of human AM/AMBP-1 may be a useful approach for attenuating organ injury, and reducing mortality after hemorrhagic shock.     

Kupffer cell cytokines interleukin-1beta and interleukin-10 combine to inhibit phosphoenolpyruvate carboxykinase and gluconeogenesis in cultured hepatocytes

BACKGROUND AND AIMS: Recent evidence suggests that inflammatory cytokines may mediate reduced hepatic glucose production and reduced blood glucose concentrations in sepsis. Therefore the aim of this study is to provide direct evidence of a cytokine-mediated interaction between Kupffer cells and hepatocytes by characterising the effects of lipopolysaccharide-stimulated Kupffer cells on hepatocyte gluconeogenesis, and the activity of key regulatory enzymes of this pathway. METHODS AND RESULTS: Primary isolates of hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells in Transwell inserts showed a 48% inhibition of gluconeogenesis (P < 0.001). RNase protection assay and ELISA of Kupffer cells and the culture media following exposure to lipopolysaccharide showed increased levels of interleukin-1 alpha and beta, tumour necrosis factor alpha and IL-10. The addition of IL-1beta and IL-10 to hepatocyte cultures inhibited gluconeogenesis by 52% (P < 0.001), whereas each cytokine alone was ineffective. To determine whether altered production or activity of phosphoenolpyruvate carboxykinase or pyruvate kinase was responsible for the reduced glucose synthesis, their mRNA, protein levels and enzyme activities were measured. Primary hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells or cultured with a combination of IL-1beta and IL-10 displayed reduced levels of phosphoenolpyruvate carboxykinase mRNA, protein and enzyme activity. In contrast the mRNA, protein levels and enzyme activity of pyruvate kinase were not altered; suggesting that gluconeogenesis was suppressed by downregulation of phosphoenolpyruvate carboxykinase. CONCLUSIONS: Therefore, hypoglycaemia, which is often observed in sepsis, may be mediated by Kupffer cell-derived IL-1beta and IL-10. In addition this study suggests these cytokines inhibit phosphoenolpyruvate carboxykinase production and thereby hepatic gluconeogenesis.     

Combination of Adrenomedullin with Its Binding Protein Accelerates Cutaneous Wound Healing

Cutaneous wound continues to cause significant morbidity and mortality in the setting of diseases such as diabetes and cardiovascular diseases. Despite advances in wound care management, there is still an unmet medical need exists for efficient therapy for cutaneous wound. Combined treatment of adrenomedullin (AM) and its binding protein-1 (AMBP-1) is protective in various disease conditions. To examine the effect of the combination treatment of AM and AMBP-1 on cutaneous wound healing, full-thickness 2.0-cm diameter circular excision wounds were surgically created on the dorsum of rats, saline (vehicle) or AM/AMBP-1 (96/320 μg kg BW) was topically applied to the wound daily and wound size measured. At days 3, 7, and 14, skin samples were collected from the wound sites. AM/AMBP-1 treated group had significantly smaller wound surface area than the vehicle group over the 14-day time course. At day 3, AM/AMBP-1 promoted neutrophil infiltration (MPO), increased cytokine levels (IL-6 and TNF-α), angiogenesis (CD31, VEGF and TGFβ-1) and cell proliferation (Ki67). By day 7 and 14, AM/AMBP-1 treatment decreased MPO, followed by a rapid resolution of inflammation characterized by a decrease in cytokines. At the matured stage, AM/AMBP-1 treatment increased the alpha smooth muscle actin expression (mature blood vessels) and Masson-Trichrome staining (collagen deposition) along the granulation area, and increased MMP-9 and decreased MMP-2 mRNA expressions. TGFβ-1 mRNA levels in AM/AMBP-1 group were 5.3 times lower than those in the vehicle group. AM/AMBP-1 accelerated wound healing by promoting angiogenesis, collagen deposition and remodeling. Treatment also shortened the days to reach plateau for wound closure. Thus, AM/AMBP-1 may be further developed as a therapeutic for cutaneous wound healing.     

Endotoxin downregulates peroxisome proliferator-activated receptor-gamma via the increase in TNF-alpha release

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is anti-inflammatory in a cell-based system and in animal models of endotoxemia. We have shown that PPAR-gamma gene expression is downregulated in macrophages after lipopolysaccharide (LPS) stimulation. However, it remains unknown whether hepatic PPAR-gamma is altered in sepsis and, if so, whether LPS directly downregulates PPAR-gamma. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP). Hepatic tissues were harvested at 5, 10, and 20 h after CLP. PPAR-gamma gene expression and protein levels were determined by RT-PCR and Western blot analysis, respectively. The results showed that PPAR-gamma gene expression decreased at 10 and 20 h and that its proteins levels were reduced at 20 h after CLP. PPAR-gamma levels were also decreased in animals that were administered LPS. To determine the direct effects of LPS on PPAR-gamma downregulation, LPS binding agent polymyxin B (PMB) was administered intramuscularly after CLP. The administration of PMB significantly reduced plasma levels of endotoxin, but it did not prevent the downregulation of PPAR-gamma expression. We found that circulating levels of TNF-alpha still remained significantly elevated in PMB-treated septic animals. We, therefore, hypothesize that the decrease of PPAR-gamma expression is TNF-alpha dependent. To investigate this, Kupffer cells (KCs) were isolated from normal rats and stimulated with LPS or TNF-alpha. TNF-alpha significantly attenuated PPAR-gamma gene expression in KCs. Although LPS decreased PPAR-gamma in KCs, the downregulatory effect of LPS was blocked by the addition of TNF-alpha-neutralizing antibodies. Furthermore, the administration of TNF-alpha-neutralizing antibodies to animals before the onset of sepsis prevented the downregulation of PPAR-gamma in sepsis. We, therefore, conclude that LPS downregulates PPAR-gamma expression during sepsis via an increase in TNF-alpha release.     

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.     

Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.     

Chemical hypoxia induces pro-inflammatory signals in fat-laden hepatocytes and contributes to cellular crosstalk with Kupffer cells through extracellular vesicles

BackgroundObstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD.MethodsPrimary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo.ResultsHypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1β, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1.ConclusionHypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].