Direct Cytoprotective Effects Research Articles

Tocotrienols Provide Radioprotection to Multiple Organ Systems through Complementary Mechanisms of Antioxidant and Signaling Effects.

Tocotrienols have powerful radioprotective properties in multiple organ systems and are promising candidates for development as clinically effective radiation countermeasures. To facilitate their development as clinical radiation countermeasures, it is crucial to understand the mechanisms behind their powerful multi-organ radioprotective properties. In this context, their antioxidant effects are recognized for directly preventing oxidative damage to cellular biomolecules from ionizing radiation. However, there is a growing body of evidence indicating that the radioprotective mechanism of action for tocotrienols extends beyond their antioxidant properties. This raises a new pharmacological paradigm that tocotrienols are uniquely efficacious radioprotectors due to a synergistic combination of antioxidant and other signaling effects. In this review, we have covered the wide range of multi-organ radioprotective effects observed for tocotrienols and the mechanisms underlying it. These radioprotective effects for tocotrienols can be characterized as (1) direct cytoprotective effects, characteristic of the classic antioxidant properties, and (2) other effects that modulate a wide array of critical signaling factors involved in radiation injury.

Isolation of High-Purity Betanin from Red Beet and Elucidation of Its Antioxidant Activity against Peroxynitrite: An in vitro Study.

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO-) is the most toxic molecular species among RNS. Betalains are known to possess ONOO- scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO- reaction, ONOO--dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO- cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO- toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.

Mechanisms of enhanced renal and hepatic erythropoietin synthesis by sodium-glucose cotransporter 2 inhibitors.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of major heart failure events, an action that is statistically linked to enhanced erythropoiesis, suggesting that stimulation of erythropoietin and cardioprotection are related to a shared mechanism. Four hypotheses have been proposed to explain how these drugs increase erythropoietin production: (i) renal cortical reoxygenation with rejuvenation of erythropoietin-producing cells; (ii) counterregulatory distal sodium reabsorption leading to increased tubular workload and oxygen consumption, and thus, to localized hypoxia; (iii) increased iron mobilization as a stimulus of hypoxia-inducible factor-2α (HIF-2α)-mediated erythropoietin synthesis; and (iv) direct HIF-2α activation and enhanced erythropoietin gene transcription due to increased sirtuin-1 (SIRT1) signaling. The first two hypotheses assume that the source of increased erythropoietin is the interstitial fibroblast-like cells in the deep renal cortex. However, SGLT2 inhibitors do not alter regional tissue oxygen tension in the non-diabetic kidney, and renal erythropoietin synthesis is markedly impaired in patients with anemia due to chronic kidney disease, and yet, SGLT2 inhibitors produce an unattenuated erythrocytic response in these patients. This observation raises the possibility that the liver contributes to the production of erythropoietin during SGLT2 inhibition. Hypoxia-inducible factor-2α and erythropoietin are coexpressed not only in the kidney but also in hepatocytes; the liver is a major site of production when erythropoietin stimulation is maintained for prolonged periods. The ability of SGLT2 inhibitors to improve iron mobilization by derepressing hepcidin and ferritin would be expected to increase cytosolic ferrous iron, which might stimulate HIF-2α expression in both the kidney and liver through the action of iron regulatory protein 1. Alternatively, the established ability of SGLT2 inhibitors to enhance SIRT1 might be the mechanism of enhanced erythropoietin production with these drugs. In hepatic cell lines, SIRT1 can directly activate HIF-2α by deacetylation, and additionally, through an effect of SIRT in the liver, peroxisome proliferator-activated receptor-γ coactivator-1α binds to hepatic nuclear factor 4 to promote transcription of the erythropoietin gene and synthesis of erythropoietin. Since SIRT1 up-regulation exerts direct cytoprotective effects on the heart and stimulates erythropoietin, it is well-positioned to represent the shared mechanism that links erythropoiesis to cardioprotection during SGLT2 inhibition.

Co-Transplant of Parathyroid Gland and Stem Cell-Derived Insulin-Producing Cells Enhances Graft Survival through Release of Pro-Angiogenic and Pro-Survival Factors

Introduction Ischemia dramatically impairs survival and function of islets after transplantation. Human Stem Cell-derived Beta Cells are currently being tested in clinical trials for replacement therapy. Although stem cell technology represents an unlimited source of beta cells, it is paramount to reduce the effects of ischemia to optimize outcome. Parathyroid glands (PTG) are known to engraft in the subcutaneous and intra-muscular sites with high success rate. The objective of this study is to investigate if PTG can protect beta-cell grafts. We hypothesize that PTG protect beta cells by both accelerating neo-vasculogenesis and by producing tropic factors that sustain beta cell viability before revascularization. Methods Stem Cell-derived Beta Cells were produced from a human embryonic stem cell line with a GFP transgene knocked into the insulin locus and a constitutively expressed luciferase transgene. Insulin-producing GFP+ cells were enriched by FACS and reaggregated to form enhanced beta clusters (eBCs). eBC were transplanted under the subcutaneous space (SQ) of NSG mice and graft mass was monitored using bioluminescence imaging. Human PTG were finely cut and co-transplanted with eBC. PTG-derived CD34+ and CD34- cells were FACS purified, cultured for 24 hours and supernatants were assessed for cytokines using Luminex. Effects of PTG-derived factors on eBCs viability after challenges of hypoxia and nutrient deprivation were assessed in vitro. Results and Discussion eBC graft mass decreased by 60% in two weeks after transplant under the SQ. However, when eBC grafts were co-transplanted with PTG, bioluminescent signal increased up to 150% (Figure 1 A and B). We further investigated the mechanisms of PTG protection. We found PTG-derived CD34+ cells produced CCL2, CXCL12, VEGF-A, and PDGF-AA (Figure 2), which have all been previously implicated in neo-vasculogenesis. Consistently, PTG co-transplant led to a dramatic increase in the ingrowth of CD31+ cells. Additionally, PTG secreted parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and γ-aminobutyric acid (GABA). These factors improved eBC survival during in vitro-simulated ischemia challenge using nutrient-deprivation and/or hypoxia (Figure 3). Conclusions Our results show that PTG co-transplantation preserves beta cell graft survival. PTG-derived CD34+ cells promote neovascularization of eBC grafts by producing angiogenic factors. PTG may also have direct cytoprotective effects on eBCs by producing PTH, PTHrP, and GABA. These properties of PTG may be harnessed to minimize ischemic injury to eBC after transplant to realize the therapeutic potential of stem cell-based therapy for diabetes.

Involvement of estrogen receptors in silibinin protection of pancreatic β-cells from TNFα- or IL-1β-induced cytotoxicity

Silibinin is a polyphenolic flavonoid that exhibits anticarcinogenic, anti-inflammatory and cytoprotective effects. The effect of silibinin on pancreatic islet β-cell is yet largely unknown in spite of well documented-hepatoprotective effects. Protecting the functional insulin-producing β-cells in the pancreas is a major therapeutic challenge in the patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). This study reports the effect of silibinin on the rat pancreatic β-cell line, INS-1, damaged with pro-inflammatory cytokine, TNFα or IL-1β. Exposure to TNFα or IL-1β for 48 h caused INS-1 cells to reduce the production of insulin as well as cell viability. These actions of TNFα or IL-1β are associated with suppression of the expression of estrogen receptors (ERs). Further study revealed that silibinin protected the suppression in the expression of both ERα and ERβ that were involved in insulin synthesis and release, respectively. Furthermore, evidence is obtained that silibinin may impede the loss of critical INS-1 cells by promoting autophagy and preventing apoptosis. Direct cytoprotective effects of silibinin on INS-1 cells suggest that silibinin may be therapeutically beneficial for diabetes.

SanggenonC protects against cardiomyocyte hypoxia injury by increasing autophagy.

Sanggenon C is isolated from Morus alba, a plant that has been used for anti-inflammatory purposes in Oriental medicine. Little is known about the effect of Sanggenon C on cardiomyocyte hypoxia injury. This study, using H9c2 rat cardiomyoblasts, was designed to determine the effects of Sanggenon C on cardiomyocyte hypoxia injury. Inflammatory cytokine levels were measured by reverse transcription-polymerase chain reaction, reactive oxygen species were measured by 2′,7′-dichlorofluorescin diacetate fluorescent probe, autophagy was detected using the LC3II/I ratio and cell apoptosis was detected by TUNEL staining. The molecular mechanisms underlying Sanggenon C-induced cyto-protection were also determined by western blotting, especially the possible involvement of autophagy and AMP-activated protein kinase (AMPK). Results indicated that samples pretreated with different concentrations of Sanggenon C (1, 10 and 100 µM) reduced the expression levels of pro-inflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-1 and IL-6, under hypoxia. The beneficial effects of Sanggenon C were also associated with reduced levels of reactive oxygen species generation and increased levels of antioxidant nitric oxide and superoxide dismutase. Sanggenon C enhanced hypoxia-induced autophagy as evidenced by the increased expression levels of autophagy-associated proteins Beclin and autophagy related 5 as well as the decreased the accumulation of p62, and increased the LC3II/I ratio. Sanggenon C also reduced hypoxia-induced apoptosis as detected by TUNEL staining and the expression of Bcl-2 proteins. The beneficial effects of Sanggenon C were associated with enhanced activation level of AMPKα and suppressed hypoxia-induced mechanistic target of rapamycin (mTOR) and forkhead box O3a (FOXO3a) phosphorylation. The AMPK inhibitor Compound C (CpC) was used, and the anti-apoptotic and pro-autophagy effects of Sanggenon C in response to hypoxia were abolished by CpC. In conclusion, the current study demonstrated that Sanggenon C possessed direct cytoprotective effects against hypoxia injury in cardiac cells via signaling mechanisms involving the activation of AMPK and concomitant inhibition of mTOR and FOXO3a.

Protective effect of ursodeoxycholic acid against chemotherapy-induced mucositis.

796 Background: Gastrointestinal mucositis is a serious side effect of chemotherapy. It increases the frequency of infection, risk of bleeding, and duration of hospitalization, consequently reducing subsequent chemotherapy doses. Ursodeoxycholic acid (UDCA), which is currently used in various liver diseases, exerts direct cytoprotective effects by stabilizing membranes, inhibiting apoptosis, and acting as an antioxidant. The protective effect of UDCA against chemotherapy-induced mucositis was assessed using an in vivo animal model. Methods: Sprague-Dawley rats were randomly assigned to the following 5 groups: non-chemotherapy and vehicle; 5-fluorouracil (5-FU) and vehicle; 5-FU and 10 mg/kg/day UDCA; 5-FU and 100 mg/kg/day UDCA; and 5-FU and 500 mg/kg/day UDCA. 5-FU (400 mg/kg) or physiological saline (control) was administered by intraperitoneal injection. UDCA was orally administered 1 day before 5-FU injection for 6 days. One day after the final UDCA dose, rats were sacrificed, and the intestines were dissected for tissue sampling and laboratory analysis. Results: UDCA promoted a higher body weight recovery, decreased villus destruction, and reduced inflammatory cytokines levels, at doses of 10 and 100 mg/kg/day. Villous fusion and destruction were pronounced in the 5-FU group compared with those observed in the UDCA-treated group or controls. The jejunal villous lengths were as follows: 212.8±58.0 µm, 331.3±18.0 µm, and 310.0±112.6 µm, in the 5-FU and vehicle, 5-FU and 10 mg/kg UDCA (p = 0.006), and 5-FU and 100 mg/kg UDCA groups (p = 0.046), respectively. Real-time polymerase chain reaction (RT-PCR) showed that IL-6 and TNF-α levels decreased in the 10 mg/kg and 100 mg/kg UDCA co-administration groups. Further, myeloperoxidase activity decreased in the UDCA co-administration group. Conclusions: UDCA significantly attenuated the reduction of the height of small intestinal villi and reduced inflammatory cytokine levels, thus highlighting the potential of UDCA as a preventive agent against chemotherapy-induced gastrointestinal mucositis. The specific protective mechanisms of UDCA on the gastrointestinal tract should be determined.

Metformin Protects H9C2 Cardiomyocytes from High-Glucose and Hypoxia/Reoxygenation Injury via Inhibition of Reactive Oxygen Species Generation and Inflammatory Responses: Role of AMPK and JNK.

Metformin is a first-line drug for the management of type 2 diabetes. Recent studies suggested cardioprotective effects of metformin against ischemia/reperfusion injury. However, it remains elusive whether metformin provides direct protection against hypoxia/reoxygenation (H/R) injury in cardiomyocytes under normal or hyperglycemic conditions. This study in H9C2 rat cardiomyoblasts was designed to determine cell viability under H/R and high-glucose (HG, 33 mM) conditions and the effects of cotreatment with various concentrations of metformin (0, 1, 5, and 10 mM). We further elucidated molecular mechanisms underlying metformin-induced cytoprotection, especially the possible involvement of AMP-activated protein kinase (AMPK) and Jun NH(2)-terminal kinase (JNK). Results indicated that 5 mM metformin improved cell viability, mitochondrial integrity, and respiratory chain activity under HG and/or H/R (P < 0.05). The beneficial effects were associated with reduced levels of reactive oxygen species generation and proinflammatory cytokines (TNF-α, IL-1α, and IL-6) (P < 0.05). Metformin enhanced phosphorylation level of AMPK and suppressed HG + H/R induced JNK activation. Inhibitor of AMPK (compound C) or activator of JNK (anisomycin) abolished the cytoprotective effects of metformin. In conclusion, our study demonstrated for the first time that metformin possessed direct cytoprotective effects against HG and H/R injury in cardiac cells via signaling mechanisms involving activation of AMPK and concomitant inhibition of JNK.

Epoxyeicosanoid Signaling Provides Multi-target Protective Effects on Neurovascular Unit in Rats After Focal Ischemia.

Multiple players are involved in the highly complex pathophysiologic responses after stroke. Therefore, therapeutic approaches that target multiple cellular elements of the neurovascular unit in the damage cascade hold considerable promise for the treatment of stroke. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active eicosanoids called epoxyeicosatrienoic acids (EETs), which are further converted by soluble epoxide hydrolase (sEH) to less bioactive diols. EETs have been shown to exert direct cytoprotective effects upon several individual components of the neurovascular unit under simulated ischemic conditions in vitro. However, the cellular mechanism underlying EET-mediated neuroprotective effects after ischemia remains to be clarified. In this study, we investigated the effects of 14,15-EET and 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), a selective inhibitor of sEH, on multiple elements of neurovascular unit of the rat brain after middle cerebral artery occlusion-induced focal ischemia. The results showed that exogenous administration of 14,15-EET or AUDA could suppress astrogliosis and glial scar formation, inhibit microglia activation and inflammatory response, promote angiogenesis, attenuate neuronal apoptosis and infarct volume, and further promote the behavioral function recovery after focal ischemia. The results suggest that epoxyeicosanoid signaling is a promising multi-mechanism therapeutic target for the treatment of stroke.

Cytoprotective-selective activated protein C therapy for ischaemic stroke.

Despite years of research and efforts to translate stroke research to clinical therapy, ischaemic stroke remains a major cause of death, disability, and diminished quality of life. Primary and secondary preventive measures combined with improved quality of care have made significant progress. However, no novel drug for ischaemic stroke therapy has been approved in the past decade. Numerous studies have shown beneficial effects of activated protein C (APC) in rodent stroke models. In addition to its natural anticoagulant functions, APC conveys multiple direct cytoprotective effects on many different cell types that involve multiple receptors including protease activated receptor (PAR) 1, PAR3, and the endothelial protein C receptor (EPCR). Application of molecular engineered APC variants with altered selectivity profiles to rodent stroke models demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. Extensive basic, preclinical, and clinical research provided a compelling rationale based on strong evidence for translation of APC therapy that has led to the clinical development of the cytoprotective-selective APC variant, 3K3A-APC, for ischaemic stroke. Recent identification of non-canonical PAR1 and PAR3 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signalling as opposed to the canonical signalling provides a mechanistic explanation for how APC-mediated PAR activation can selectively induce cytoprotective signalling pathways. Collectively, these paradigm-shifting discoveries provide detailed insights into the receptor targets and the molecular mechanisms for neuroprotection by cytoprotective-selective 3K3A-APC, which is currently a biologic drug in clinical trials for ischaemic stroke.

Cardioprotective effects of exenatide against oxidative stress-induced injury

Myocardial ischemia/reperfusion (MI/R) leads to oxidative stress, which may in turn lead to myocardial injury. In the present study, we investigated the effects of exenatide, a glucagon-like peptide-1 (GLP-1) analogue, on oxidative stress-induced injury in vitro and in vivo. In in vitro experiments, H9c2 cells were incubated with exenatide to determine the direct cytoprotective effects of exenatide following exposure to hydrogen peroxide (H2O2). Pre-treatment with exenatide (1 nM), prior to H2O2 exposure, increased cell viability and inhibited H2O2-induced reactive oxygen species (ROS) production. Exenatide also decreased the levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in the cultured supernatants, as well as those of malondialdehyde (MDA) in the H9c2 cells and increased the total superoxide dismutase (T-SOD) levels in the H9c2 cells. In in vivo experiments, an animal model of MI/R was induced by coronary occlusion. Pre-treatment with exenatide (10 µg/kg/day) protected the rat hearts from MI/R-induced injury by decreasing the levels of LDH and CK-MB in plasma, increasing the levels of catalase, T-SOD and glutathione peroxidase (GSH-Px) in the heart and decreasing the MDA levels in the rats with MI/R-induced injury. Exenatide also reduced the infarct size and enhanced cardiac function in the rats with MI/R-induced injury. Moreover, pre-treatment with exenatide inhibited cardiomyocyte apoptosis, increased Aktserine473 and Badserine136 phosphorylation and decreased cleaved caspase-3 expression in vitro and in vivo; however, these effects were attenuated by the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. Our results suggest that exenatide exerts significant cardioprotective effects against oxidative stress-induced injury in vitro and in vivo. The mechanisms involved may be attributed to the scavenging of oxidative stress products, such as ROS, the increase in the concentrations of antioxidant defense enzymes and the inhibition of cardiomyocyte apoptosis. The anti-apoptotic effects of exenatide were, at least in part, associated with the activation of the PI3K/Akt signaling pathway.

Activated Protein C Inhibits Pancreatic Islet Inflammation, Stimulates T Regulatory Cells, and Prevents Diabetes in Non-obese Diabetic (NOD) Mice

Activated protein C (aPC) is a natural anticoagulant with strong cyto-protective and anti-inflammatory properties. aPC inhibits pancreatic inflammation and preserves functional islets after intraportal transplantation in mice. Whether aPC prevents the onset or development of type 1 diabetes (T1D) is unknown. In this study, when human recombinant aPC was delivered intraperitoneally, twice weekly for 10 weeks (from week 6 to 15) to non-obese diabetic (NOD) mice, a model for T1D, the incidence of diabetes was reduced from 70% (saline control) to 7.6% by 26 weeks of age. Islets of aPC-treated mice exhibited markedly increased expression of insulin, aPC/protein C, endothelial protein C receptor, and matrix metalloproteinase (MMP)-2 when examined by immunostaining. The insulitis score in aPC-treated mice was 50% less than that in control mice. T regulatory cells (Tregs) in the spleen, pancreatic islets, and pancreatic lymph nodes were increased 37, 53, and 59%, respectively, in NOD mice following aPC treatment. These Tregs had potent suppressor function and, after adoptive transfer, delayed diabetes onset in NOD.severe combined immunodeficiency mice. The culture of NOD mouse spleen cells with aPC reduced the secretion of inflammatory cytokines interleukin (IL)-1β and interferon-γ but increased IL-2 and transforming growth factor-β1, two cytokines required for Treg differentiation. In summary, our results indicate that aPC prevents T1D in the NOD mouse. The aPC mechanism of action is complex, involving induction of Treg differentiation, inhibition of inflammation, and possibly direct cyto-protective effects on β cells.

Klotho protects against oxidative damage via amelioration of phosphotoxicity

Klotho mitigates aging in various organs via a host of mechanisms including anti‐oxidation and anti‐phosphotoxicity. Organ damage in Klotho‐deficient animals can be rescued by dietary phosphate (Pi) restriction. The pulmonary epithelium is exposed to constant environmental insults. Klotho‐deficient animals exhibit alveolar airspace enlargement suggesting accelerated aging. To test the direct effects of Klotho against O2 and Pi toxicity in vitro, we exposed A549 lung epithelial cells to hyperoxia (95‐5% O2‐CO2) and increasing Pi (1, 3, 5 mM). Soluble (truncated) or transmembrane (full length) Klotho was over‐expressed by transfection. Apoptosis was measured from caspase‐8 activity (Table, nmol.[hr.104 cells]−1, mean±SD).Both hyperoxia and Pi induce apoptosis in A549 cells; the effects are synergistic (p&lt;0.05, repeated measures ANOVA with respect to O2 and Pi). Both transmembrane and soluble Klotho overexpression markedly reduced cell apoptosis induced by Pi loading especially in hyperoxia. We conclude that Klotho exerts direct cytoprotective effects on pulmonary epithelium via anti‐oxidative and anti‐phosphotoxic effects.Support: F32 HL103043, RO1 DK091392, DK092461

Hematopoietic cytokines for cardiac repair: mobilization of bone marrow cells and beyond

Hematopoietic cytokines, traditionally known to influence cellular proliferation, differentiation, maturation, and lineage commitment in the bone marrow, include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, stem cell factor, Flt-3 ligand, and erythropoietin among others. Emerging evidence suggests that these cytokines also exert multifarious biological effects on diverse nonhematopoietic organs and tissues. Although the precise mechanisms remain unclear, numerous studies in animal models of myocardial infarction (MI) and heart failure indicate that hematopoietic cytokines confer potent cardiovascular benefits, possibly through mobilization and subsequent homing of bone marrow-derived cells into the infarcted heart with consequent induction of myocardial repair involving multifarious mechanisms. In addition, these cytokines are also known to exert direct cytoprotective effects. However, results from small-scale clinical trials of G-CSF therapy as a single agent after acute MI have been discordant and largely disappointing. It is likely that cardiac repair following cytokine therapy depends on a number of known and unknown variables, and further experimental and clinical studies are certainly warranted to accurately determine the true therapeutic potential of such therapy. In this review, we discuss the biological features of several key hematopoietic cytokines and present the basic and clinical evidence pertaining to cardiac repair with hematopoietic cytokine therapy.

The structure-function relationship of activated protein C

Protein C is the central enzyme of the natural anticoagulant pathway and its activated form APC (activated protein C) is able to proteolyse non-active as well as active coagulation factors V and VIII. Proteolysis renders these cofactors inactive, resulting in an attenuation of thrombin formation and overall down-regulation of coagulation. Presences of the APC cofactor, protein S, thrombomodulin, endothelial protein C receptor and a phospholipid surface are important for the expression of anticoagulant APC activity. Notably, APC also has direct cytoprotective effects on cells: APC is able to protect the endothelial barrier function and expresses anti-inflammatory and anti-apoptotic activities. Exact molecular mechanisms have thus far not been completely described but it has been shown that both the protease activated receptor 1 and EPCR are essential for the cytoprotective activity of APC. Recently it was shown that also other receptors like sphingosine 1 phosphate receptor 1, Cd11b/CD18 and tyrosine kinase with immunoglobulin-like and EGF-like domains 2 are likewise important for APC signalling. Mutagenesis studies are being performed to map the various APC functions and interactions onto its 3D structure and to dissect anticoagulant and cytoprotective properties. The results of these studies have provided a wealth of structure-function information. With this review we describe the state-of-the-art of the intricate structure-function relationships of APC, a protein that harbours several important functions for the maintenance of both humoral and tissue homeostasis.

G-CSF and Erythropoietin Combination Therapy for Infarct Repair: Two Plus Two Equals Two?

G-CSF and Erythropoietin Combination Therapy for Infarct Repair: Two Plus Two Equals Two?

Activated protein C analog with reduced anticoagulant activity improves functional recovery and reduces bleeding risk following controlled cortical impact

The anticoagulant activated protein C (APC) protects neurons and vascular cells from injury through its direct cytoprotective effects that are independent of its anticoagulant action. Wild-type recombinant murine APC (wt-APC) exerts significant neuroprotection in mice if administered early after traumatic brain injury (TBI). Here, we compared efficacy and safety of a late therapy for TBI with wt-APC and 3K3A-APC, an APC analog with ∼ 80% reduced anticoagulant activity but normal cytoprotective activity, using a controlled cortical impact model of TBI. Mice received 0.8 mg/kg intraperitoneally of recombinant murine 3K3A-APC, wt-APC or saline at 6, 12, 24 and 48 h after injury. 3K3A-APC ( n = 15) relative to wt-APC ( n = 15) improved motor and sensorimotor recovery within the first three days post-trauma as demonstrated by rotarod ( p < 0.05) and beam balance test ( p < 0.05), respectively. Both, wt-APC and 3K3A-APC reduced the lesion volume seven days after injury by 36% ( n = 8; p < 0.01) and 56% ( n = 8; p < 0.01), respectively, compared to saline ( n = 8). Three days post-TBI, the hemoglobin levels in the injured brain were increased by ∼ 3-fold after wt-APC treatment compared to saline indicating an increased risk for intracerebral bleeding. In contrast, comparable levels of brain hemoglobin in 3K3A-APC-treated and saline-treated mice suggested that 3K3A-APC treatment did not increase risk for bleeding after TBI. Thus, compared to wt-APC, 3K3A-APC is more efficacious and safer therapy for TBI with no risk for intracerebral hemorrhage.

Heparin-binding EGF-like growth factor is a potent dilator of terminal mesenteric arterioles

Objective We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) protects the intestines from multiple forms of injury via direct cytoprotective effects on the intestinal mucosa. In this study, we examined the effects of HB-EGF on the hemodynamics of intestinal arterioles, the major resistance vessels that regulate blood flow to the intestines, as an additional mechanism of HB-EGF-mediated intestinal protection. Methods The hemodynamic effects of HB-EGF in rodent terminal mesenteric arterioles and human submucosal arterioles were examined ex vivo using a video dimension analyzer. Cultured human intestinal microvascular endothelial cells (HIMEC) were used to elucidate the mechanisms of HB-EGF-induced vasodilation. Results HB-EGF significantly increased vessel diameter under conditions of increasing intraluminal pressure and increased flow rate. These HB-EGF-mediated vasodilatory effects were observed in terminal mesenteric arterioles from adult rats and 3 day old rat pups. These effects were confirmed in submucosal arterioles from human intestine. Furthermore, HB-EGF significantly reduced endothelin-1-induced mesenteric arteriolar vasoconstriction. The vasodilatory effects of HB-EGF were blocked by ET B receptor antagonism in adult rat arterioles, and also by nitric oxide synthase inhibition in rat pup and human infant arterioles. In HIMEC, HB-EGF significantly increased endothelin B (ET B) receptor protein expression and provoked intracellular calcium mobilization. Conclusions HB-EGF is a potent vasodilator of the intestinal microvasculature, further supporting its use in diseases manifested by decreased intestinal blood flow, including necrotizing enterocolitis.

Resveratrol protects cardiomyocytes from hypoxia-induced apoptosis through the SIRT1–FoxO1 pathway

Loss of cardiomyocytes through apoptosis has been proposed as a cause of ventricular remodeling and heart failure. Ischemia- and hypoxia-induced apoptosis of cardiomyocytes reportedly plays an important role in many cardiac pathologies. We investigated whether resveratrol (Res) has direct cytoprotective effects against ischemia/hypoxia for cardiomyocytes. Exposure of H9c2 embryonic rat heart-derived cells to hypoxia for 24 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry, while treatment with 20 μM Res greatly decreased hypoxia-induced apoptosis in these cells. Exposure of the cells to Res (20 μM) caused rapid activation of SIRT1, which had a dual effect on FoxO1 function: SIRT1 increased FoxO1’s ability to induce cell cycle arrest, but inhibited FoxO1’s ability to induce cell death. This effect could be reversed by SIRT1 inhibition. Results of our study indicate that Res inhibits hypoxia-induced apoptosis via the SIRT1–FoxO1 pathway in H9c2 cells. This polyphenol may have potential in preventing cardiovascular disease, especially in coronary artery disease (CAD) patients.

G-CSF- and erythropoietin-based cell therapy: a promising strategy for angiomyogenesis in myocardial infarction

Granulocyte colony-stimulating factor (G-CSF) and erythropoietin are two cytokines that have been demonstrated to improve cardiac function and perfusion in myocardial infarction. G-CSF was initially evaluated as a stem cell mobilizer and erythropoietin as a cytoprotective agent. However, both cytokines have direct cytoprotective effects and stem cell-mobilizing ability. Direct cytoprotective effects of both cytokines are commonly mediated by the Jak–STAT pathway. In preclinical study, G-CSF and erythropoietin improved cardiac function and perfusion by angiomyogenesis and protection of cardiomyocytes in myocardial infarction. However, results from recent clinical trials did not support beneficial effects of cytokine therapy with G-CSF or erythropoietin alone in patients with myocardial infarction. Further studies are required to elucidate the mechanism of action and to improve therapeutic efficacy by employing novel strategies, such as combined cytokines.