adrenomedullin and Ischemia

Topics: Adrenomedullin; Animals; Edema; Endothelium, Vascular; Homeostasis; Humans; Ischemia; Mice; Mice, Knockout; Molecular Targeted Therapy; Neovascularization, Physiologic; Receptor Activity-Modifying Protein 2

Adrenomedullin (AM) is a novel vasodilator peptide first purified from human pheochromocytoma by tracing its capacity to stimulate cAMP production in platelets. AM immunoreactivity is widely distributed in the central nervous system (CNS) and in the rat has been demonstrated by immunohistochemical techniques to be present in many neurons throughout the brain and spinal cord, as well as in some vascular endothelial cells and perivascular glial cells. Electron microscopy shows that the immunoreactivity is located mainly in the neuronal cytoplasm, but also occurs in the cell nucleus in some cells of the caudate putamen and olfactory tubercle. Biochemical analyses suggest that higher molecular forms, presumably precursor forms, may predominate over fully processed AM in some brain areas. The expression of AM immunoreactivity is increased in cortical neurons, endothelial cells, and perivascular processes after a simulation of ischemia by oxygen and glucose deprivation. Immunohistochemical, electrophysiological, and pharmacological studies suggest that AM in the CNS can act as a neurotransmitter, neuromodulator, or neurohormone, or as a cytoprotective factor in ischemic/hypoxic conditions, in addition to its vasodilator role.

Topics: Adrenomedullin; Animals; Brain; Humans; Hypoxia; Immunohistochemistry; Ischemia; Mice; Microscopy, Electron; Peptides; Rats; Spinal Cord

Preeclampsia is a heterogeneous hypertensive disorder of pregnancy. It affects multiorgans and may lead to fetal growth restriction, organ failure, seizure, and maternal death. Unfortunately, current treatments are ineffective at delaying the progression of preeclampsia even for a few days. Clinicians are often forced to deliver preterm fetus if severe preeclampsia occurred early during pregnancy, leading to premature birth-associated complications. Preeclampsia has been associated with defects at the maternal-fetal interface and maternal vascular dysfunction. Of interest, the adrenomedullin peptide and its cognate receptors, calcitonin receptor-like receptor (CLR)/ receptor activity-modifying protein (RAMP) receptor complexes, have been shown to be important regulators of cardiovascular adaptation and feto-placental development during pregnancy. Although the exact role of adrenomedullin-CLR/RAMP signaling in different feto-maternal compartments during pregnancy and how adrenomedullin expression affects preeclampsia development remains to be clarified, we hypothesized that the sustained activation of CLR/RAMP receptors could be a promising strategy to mitigate placental ischemia-associated vascular dysfunction and fetal growth restriction under preeclampsia-like conditions.. To explore this possibility, we have developed a stable adrenomedullin analog, ADE101, and investigated its effects on human lymphatic microvascular endothelial (HLME) cell proliferation, hemodynamics, and pregnancy outcomes in pregnant rats with reduced uteroplacental perfusion pressure (RUPP) induced by clipping of uterine arteries on gestation day 14.. The ADE101 analog has a potent effect on CLR/RAMP2 receptor activation, and an enhanced stimulatory effect on HLME cell proliferation compared to wild-type peptides. ADE101 also exhibits a lasting effect on hemodynamics in normal and hypertensive rats. In addition, studies using the RUPP model showed that ADE101 significantly reduces placental ischemia-induced hypertension and fetal growth restriction in a dose-dependent manner. Infusion of ADE101 increased the weight of fetuses and placentas in RUPP animals to 252% and 202% of that of RUPP controls, respectively.. These data suggested that long-acting adrenomedullin analog could be useful for quenching hypertension as well as the vascular ischemia-associated organ damages in preeclamptic patients.

Topics: Adrenomedullin; Animals; Blood Pressure; Female; Fetal Growth Retardation; Humans; Hypertension; Ischemia; Placenta; Pre-Eclampsia; Pregnancy; Rats; Rats, Sprague-Dawley; Uterus

A combination of vascular pathologies and other complicating factors results in chronic wounds which constitute a serious burden for both patients and national health systems, due to prolonged hospital stays, high costs, and prolonged nursing staff dedication. Here we investigate whether proadrenomedullin N-terminal 20 peptide (PAMP), a naturally occurring peptide of the skin with antimicrobial and proangiogenic properties, either alone or in combination with autologous skeletal muscle stem/progenitor cells, acts as a wound healing factor. The rabbit ear was chosen as a test system, since it offers a reliable model for normoxic and ischemic wounds. Topical treatments with PAMP, stem/progenitor cells, or a combination of both, resulted in significant improvements of healing, when compared to untreated wounds. PAMP was very effective in promoting reepithelialization and angiogenesis, whereas treatment with stem/progenitor cells alone resulted in less wound contraction. Interestingly, the combination of PAMP and stem/progenitor cells, while maintaining angiogenic potency, reverted to the contraction levels observed in the untreated controls. Under ischemic conditions, generalized necrosis of the dermis and the underlying cartilage was observed in untreated wounds. Treatments of these wounds with PAMP or stem/progenitor cells allowed a timely recovery. In conclusion, PAMP either alone or in combination with autologous stem/progenitor cells may provide a useful tool for improving wound healing both in normoxic and ischemic conditions.

Topics: Adrenomedullin; Angiogenesis Inducing Agents; Animals; Ischemia; Male; New Zealand; Rabbits; Stem Cell Transplantation; Transplantation, Autologous; Wound Healing

Leakage from colonic anastomosis is a major complication causing increased mortality and morbidity. Ischemia is a well-known cause of this event. This study was designed to investigate the effects of adrenomedullin on the healing of ischemic colon anastomosis in a rat model.. Standardized left colon resection 3 cm above the peritoneal reflection and colonic anastomosis were performed in 40 Wistar rats that were divided into four groups. To mimic ischemia, the mesocolon was ligated 2 cm from either side of the anastomosis in all of the groups. The control groups (1 and 2) received no further treatment. The experimental groups (3 and 4) received adrenomedullin treatment. Adrenomedullin therapy was started in the perioperative period in group 3 and 4 rats (the therapeutic groups). Group 1 and group 3 rats were sacrificed on postoperative day 3. Group 2 and group 4 rats were sacrificed on postoperative day 7. After careful relaparotomy, bursting pressure, hydroxyproline, malondialdehyde, interleukin 6, nitric oxide, vascular endothelial growth factor, and tumor necrosis factor alpha levels were measured. Histopathological characteristics of the anastomosis were analyzed.. The group 3 animals had a significantly higher bursting pressure than group 1 (p<0.05). Hydroxyproline levels in group 1 were significantly lower than in group 3 (p<0.05). The mean bursting pressure was significantly different between group 2 and group 4 (p<0.05). Hydroxyproline levels in groups 3 and 4 were significantly increased by adrenomedullin therapy relative to the control groups (p<0.05). When all groups were compared, malondialdehyde and nitric oxide were significantly lower in the control groups (p<0.05). When vascular endothelial growth factor levels were compared, no statistically significant difference between groups was observed. Interleukin 6 and tumor necrosis factor alpha were significantly decreased by adrenomedullin therapy (p<0.05). The healing parameters and inflammatory changes (e.g., granulocytic cell infiltration, necrosis, and exudate) were significantly different among all groups (p<0.05).. Adrenomedullin had positive effects on histopathologic anastomotic healing in this experimental model of ischemic colon anastomosis.

Topics: Adrenomedullin; Anastomosis, Surgical; Anastomotic Leak; Animals; Colon; Disease Models, Animal; Female; Ischemia; Postoperative Complications; Postoperative Period; Rats; Rats, Wistar; Time Factors; Vasodilator Agents; Wound Healing

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.

Topics: Adrenomedullin; Alanine Transaminase; Animals; Aspartate Aminotransferases; Complement Factor H; Cytokines; Humans; Intestines; Ischemia; Lactic Acid; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Survival Rate; Tumor Necrosis Factor-alpha

"Ischemic tolerance" can be induced in the retina by "preconditioning" with brief periods of non-injurious retinal ischemia or systemic hypoxia. The present study was undertaken to assess whether tolerance can be induced pharmacologically by deferroxamine (DFX), an iron chelator, which promotes the expression of the transcription factor, hypoxia-inducible factor 1-alpha (HIF-1alpha), and to identify potential HIF-1alpha -induced effectors of this endogenous protective response.. ND4 Swiss-Webster mice were preconditioned with DFX (200 mg/kg, intraperitoneally) as a single dose (SDP) or as repetitive doses (RDP; 6 doses over 2 weeks) and then subjected to 30 min of retinal ischemia (by intraocular pressure elevation) 1 or 4 weeks later. Retinal layer thicknesses and cell counts were quantified 1 week after ischemia. Retinae of additional mice were obtained at various times after SDP or RDP to examine protein-level expression of HIF-1alpha and adrenomedullin (ADM), a HIF-1alpha gene target, by immunoblotting and immunohistochemistry.. Ischemia-induced injury was significantly attenuated by SDP 1 week earlier, but not when SDP occurred 4 weeks earlier. However, RDP performed 4 weeks earlier was potently neuroprotective. DFX robustly induced HIF-1alpha protein expression throughout the inner retina, and levels of HIF-1alpha protein remained significantly elevated over the 1- and 4-week periods of time between the respective SDP and RDP stimulus and the induction of retinal ischemia. Increases in ADM protein expression were evident throughout the retina following both preconditioning treatments.. DFX preconditions the retina against ischemic injury and multiple doses promote a long-lasting, ischemia-protective phenotype. The widespread and protracted elevations in HIF-1alpha protein levels and the robust expression of one of its neuroprotective, prosurvival gene targets, ADM, strongly suggest that DFX-induced preconditioning is HIF-1alpha-dependent. The ability to pharmacologically induce ischemic tolerance in the retina by a clinically well-tolerated drug underscores the potential therapeutic utility of preconditioning for retinal protection in various ischemic retinopathies.

Topics: Adrenomedullin; Animals; Deferoxamine; Dose-Response Relationship, Drug; Hypoxia-Inducible Factor 1, alpha Subunit; Iron Chelating Agents; Ischemia; Ischemic Preconditioning; Male; Mice; Retinal Vessels

Bone marrow mononuclear cell (BM-MNC) implantation (BMI) for critical severe limb ischemia especially for Buerger's disease shows excellent clinical results but the mechanism of this treatment is still unknown. In this study, we investigated the changes in serum levels of angiogenesis-related factors after BMI treatment.. Twelve patients whose BMI treatments were clinically very effective was selected out of ninteen cases, nine patients had Buerger's disease, two patients had arteriosclerosis obliterans and one had systemic sclerosis. Venous bood from femoral vein or brachial vein of the recipient limbs of these patients.. Adrenomedulin (AM), soluble vascular cell adhesion molecule-1 (sVCAM-1), and C-reactive protein (CRP) serum levels 24 h after BMI treatment were significantly increased compared with those before BMI treatment (p < 0.05). Vascular endothelial growth factor (VEGF) serum levels after BMI treatment significantly increased between 1 week and 3 months after BMI treatment (p < 0.05). Nitric oxide (NO) serum levels after BMI treatment increased significantly 2 weeks after BMI treatment (p < 0.05). There was no correlation between the numbers of implanted cells and serum levels of measured angiogenesis-related factors that were significantly increased after BMI treatment.. It was concluded that the mechanism underlying BMI treatment consists of early and late phases. The early phase involves the direct action by implanted cells, and the late phase involves indirect paracrine action. In addition, it was considered that BMI treatment is effective when we implant a sufficient level of bone marrow (600 ml) to treat severe limb ischemia.

Topics: Adrenomedullin; Adult; Aged; Arterial Occlusive Diseases; C-Reactive Protein; Convalescence; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Follow-Up Studies; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Hepatocyte Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Ischemia; Leg; Male; Middle Aged; Nitric Oxide; Peripheral Vascular Diseases; Postoperative Period; Transplantation, Autologous; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

Hypoxia-inducible factor (HIF) plays an important role in regulating gene expression in response to ischemia. Although activation of HIF-1 in muscle tissue was found during ischemia in vivo, the meaning and mechanisms in isolated cells are still incompletely understood. We studied activation of HIF-1 in skeletal muscle cells cultured in either their undifferentiated myoblast state or differentiated into myotubes. HIF-1 was activated in myoblasts and myotubes by hypoxia and simulated ischemia. Induction of adrenomedullin mRNA and, to a lesser extent, VEGF mRNA correlated well with the induction of HIF-1alpha protein in both cell types. Enzymes of glycolysis-like lactate dehydrogenase and pyruvate kinase showed upregulation of their mRNA only under hypoxic conditions but not during simulated ischemia. Phosphofructokinase mRNA showed no significant upregulation at all. Although HIF-1 was activated in myotubes during simulated ischemia, myotubes died preceded by a loss of ATP. Myoblasts survived simulated ischemia with no decrease in ATP or ATP turnover. Furthermore, pharmacological inhibition of HIF-1 hydroxylases by dimethyloxalylglycine (DMOG) increased HIF-1alpha accumulation and significantly upregulated the expression of adrenomedullin, VEGF, lactate dehydrogenase, and pyruvate kinase in myoblasts and myotubes. However, DMOG provided no protection from cell death. Our data indicate that HIF-1, although activated in myotubes during simulated ischemia, cannot protect against the loss of ATP and cell viability. In contrast, myoblasts survive ischemia and thus may play an important role during regeneration and HIF-1-induced revascularization.

Topics: Adrenomedullin; Animals; Cell Death; Cell Line; Gene Expression Regulation; Glycolysis; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemia; Lactate Dehydrogenases; Mice; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts, Skeletal; Phosphofructokinases; Pyruvate Kinase; RNA, Messenger; RNA, Small Interfering; Vascular Endothelial Growth Factor A

Adrenomedullin exerts not only vasodilatory effects, but also angiogenic effects. In the present study, we investigated the effects of adrenomedullin on collateral formation and circulating bone-marrow-derived cells after acute tissue ischaemia. Bone marrow of 8-10-week-old female C57BL/6J mice was replaced with that from GFP (green fluorescent protein) transgenic mice (GFP mice). At 8 weeks after transplantation, hindlimb ischaemia was induced by resecting the right femoral artery and a plasmid expressing human adrenomedullin (50 mug) was injected into the ischaemic muscle, followed by in vivo electroporation on a weekly basis. Overexpression of adrenomedullin significantly enhanced the blood flow recovery compared with controls (blood flow ratio, 1.0+/-0.2 compared with 0.6+/-0.3 respectively, at week 4; P<0.05) and increased capillary density in the ischaemic leg as determined by anti-CD31 immunostaining of the ischaemic muscle (567+/-40 compared with 338+/-65 capillaries/mm(2) respectively, at week 5; P<0.05). There were more GFP-positive cells in the thigh muscle of the mice injected with adrenomedullin than in that of the control mice (29.6+/-4.5 compared with 16.5+/-3.3 capillaries/mm(2) respectively, at week 5; P<0.05). We repeated the same experiments using LacZ-knock-in mice instead of GFP mice, and obtained similar results. These findings suggest that adrenomedullin may augment ischaemia-induced collateral formation with some effects on circulating bone-marrow-derived cells.

Topics: Adrenomedullin; Animals; Bone Marrow Transplantation; Collateral Circulation; Female; Flow Cytometry; Green Fluorescent Proteins; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Hindlimb; Ischemia; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Vasodilator Agents

Previous studies have shown that adrenomedullin (AM) inhibits vascular endothelial cell apoptosis and induces angiogenesis. We investigated whether AM enhances bone marrow cell-induced angiogenesis.. Immediately after hindlimb ischemia was created, rats were randomized to receive AM infusion plus bone marrow-derived mononuclear cell (MNC) transplantation (AM+MNC group), AM infusion alone (AM group), MNC transplantation alone (MNC group), or vehicle infusion (control group). The laser Doppler perfusion index was significantly higher in the AM and MNC groups than in the control group (0.74+/-0.11 and 0.69+/-0.07 versus 0.59+/-0.07, respectively, P<0.01), which suggests the angiogenic potency of AM and MNC. Importantly, improvement in blood perfusion was marked in the AM+MNC group (0.84+/-0.08). Capillary density was highest in the AM+MNC group, followed by the AM and MNC groups. In vitro, AM inhibited MNC apoptosis, promoted MNC adhesiveness to a human umbilical vein endothelial cell monolayer, and increased the number of MNC-derived endothelial progenitor cells. In vivo, AM administration not only enhanced the differentiation of MNC into endothelial cells but also produced mature vessels that included smooth muscle cells.. A combination of AM infusion and MNC transplantation caused significantly greater improvement in hindlimb ischemia than MNC transplantation alone. This effect may be mediated in part by the angiogenic potency of AM itself and the beneficial effects of AM on the survival, adhesion, and differentiation of transplanted MNCs.

Topics: Adrenomedullin; Angiogenesis Inducing Agents; Animals; Apoptosis; Bone Marrow Transplantation; Cell Adhesion; Cell Differentiation; Cell Movement; Cells, Cultured; Combined Modality Therapy; Endothelial Cells; Hindlimb; Humans; Ischemia; Male; Myocytes, Smooth Muscle; Neovascularization, Physiologic; Peptides; Peripheral Vascular Diseases; Random Allocation; Rats; Rats, Inbred Lew; Stem Cells; Umbilical Veins; Vasodilator Agents

Adrenomedullin (AM) induces angiogenesis and inhibits cell apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. Transplantation of mesenchymal stem cells (MSCs) has been shown to improve neurological deficits after stroke in rats. We investigated whether AM enhances the therapeutic potency of MSC transplantation.. Male Lewis rats (n=100) were subjected to 2-hour middle cerebral artery occlusion. Immediately after reperfusion, rats were assigned randomly to receive intravenous transplantation of MSCs plus subcutaneous infusion of AM for 7 days (MSC+AM group), AM infusion alone (AM group), MSC transplantation alone (MSC group), or vehicle infusion (control group). Neurological and immunohistological assessments were performed to examine the effects of these treatments.. Some engrafted MSCs were positive for neuronal and endothelial cell markers, although the number of differentiated MSCs did not differ significantly between the MSC and MSC+AM groups. The neurological score significantly improved in the MSC, AM, and MSC+AM groups compared with the control group. Importantly, improvement in the MSC+AM group was significantly greater than that in the MSC and AM groups. There was marked induction of angiogenesis in the ischemic penumbra in the MSC+AM group, followed by the AM, MSC, and control groups. AM infusion significantly inhibited apoptosis of transplanted MSCs. As a result, the number of engrafted MSCs in the MSC+AM group was significantly higher than that in the MSC group.. AM enhanced the therapeutic potency of MSCs, including neurological improvement, possibly through inhibition of MSC apoptosis and induction of angiogenesis.

Topics: Adrenomedullin; Animals; Apoptosis; Body Weight; Cell Transplantation; Disease Models, Animal; Immunohistochemistry; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Ischemia; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Neovascularization, Pathologic; Neurons; Peptides; Phosphatidylinositol 3-Kinases; Rats; Rats, Inbred Lew; Stem Cells; Stroke; Time Factors

To investigate regional aspects of hypoxic regulation of adrenomedullin (AM) in kidneys, we mapped the distribution of AM in the rat kidney after hypoxia (normobaric hypoxic hypoxia, carbon monoxide, and CoCl(2) for 6 h), anemia (hematocrit lowered by bleeding) and after global transient ischemia for 1 h (unilateral renal artery occlusion and reperfusion for 6 and 24 h) and segmental infarct (6 and 24 h). AM expression and localization was determined in normal human kidneys and in kidneys with arterial stenosis. Hypoxia stimulated AM mRNA expression significantly in rat inner medulla (CO 13 times, 8% O(2) 6 times, and CoCl(2) 8 times), followed by the outer medulla and cortex. AM mRNA level was significantly elevated in response to anemia and occlusion-reperfusion. Immunoreactive AM was associated with the thin limbs of Henle's loop, distal convoluted tubule, collecting ducts, papilla surface epithelium, and urothelium. AM labeling was prominent in the inner medulla after CO and in the outer medulla after occlusion-reperfusion. The infarct border zone was strongly labeled for AM. In cultured inner medullary collecting duct cells, AM mRNA was significantly increased by hypoxia. AM mRNA was equally distributed in human kidney and AM was localized as in the rat kidney. In human kidneys with artery stenosis, AM mRNA was not significantly enhanced compared with controls, but AM immunoreactivity was observed in tubules, vessels, and glomerular cells. In summary, AM expression was increased in the rat kidney in response to hypoxic and ischemic hypoxia in keeping with oxygen gradients. AM was widely distributed in the human kidney with arterial stenosis. AM may play a significant role to counteract hypoxia in the kidney.

Topics: Adrenomedullin; Anemia; Animals; Cells, Cultured; Gene Expression; Humans; Hypertension, Renal; Hypoxia; Immunohistochemistry; Ischemia; Kidney; Male; Peptides; Rats; Rats, Sprague-Dawley; Renal Artery Obstruction; RNA, Messenger

Earlier studies have shown that adrenomedullin (AM), a potent vasodilator peptide, has a variety of cardiovascular effects. However, whether AM has angiogenic potential remains unknown. This study investigated whether AM gene transfer induces therapeutic angiogenesis in chronic hind limb ischemia.. Ischemia was induced in the hind limb of 21 Japanese White rabbits. Positively charged biodegradable gelatin was used to produce ionically linked DNA-gelatin complexes that could delay DNA degradation. Human AM DNA (naked AM group), AM DNA-gelatin complex (AM-gelatin group), or gelatin alone (control group) was injected into the ischemic thigh muscles. Four weeks after gene transfer, significant improvements in collateral formation and hind limb perfusion were observed in the naked AM group and AM-gelatin group compared with the control group (calf blood pressure ratio: 0.60+/-0.02, 0.72+/-0.03, 0.42+/-0.06, respectively). Interestingly, hind limb perfusion and capillary density of ischemic muscles were highest in the AM-gelatin group, which revealed the highest content of AM in the muscles among the three groups. As a result, necrosis of lower hind limb and thigh muscles was minimal in the AM-gelatin group.. AM gene transfer induced therapeutic angiogenesis in a rabbit model of chronic hind limb ischemia. Furthermore, the use of biodegradable gelatin as a nonviral vector augmented AM expression and thereby enhanced the therapeutic effects of AM gene transfer. Thus, gelatin-mediated AM gene transfer may be a new therapeutic strategy for the treatment of peripheral vascular diseases.

Topics: Adrenomedullin; Animals; Chronic Disease; Gelatin; Gene Expression; Genetic Vectors; Ischemia; Lower Extremity; Male; Neovascularization, Physiologic; Peptides; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rabbits; Radiography

Adrenomedullin (AM) is a novel vasodilating peptide involved in the regulation of circulatory homeostasis and implicated in the pathophysiology of cardiovascular disease. We tested the hypothesis that AM also possesses angiogenic properties. Using laser Doppler perfusion imaging, we found that AM stimulated recovery of blood flow to the affected limb in the mouse hind-limb ischemia model. AM exerted this effect in part by promoting expression of vascular endothelial growth factor (VEGF) in the ischemic limb, and immunostaining for CD31 showed the enhanced flow to reflect increased collateral capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. However, heterozygotic AM knockout mice (AM+/-) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression than their wild-type littermates. Similarly, mice treated with AM22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited in AM+/- and AM22-52-treated mice. Notably, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM+/- and AM22-52-treated mice. In cocultures of endothelial cells and fibroblasts, AM enhanced VEGF-induced capillary formation, whereas in cultures of endothelial cells AM enhanced VEGF-induced Akt activation. These results show that AM possesses novel angiogenic properties mediated by its ability to enhance VEGF expression and Akt activity. This may make AM a useful therapeutic tool for relieving ischemia; conversely, inhibitors of AM could be useful for clinical management of tumor growth.

Topics: Adrenomedullin; Animals; Capillaries; Coculture Techniques; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Fibroblasts; Genetic Therapy; Hindlimb; Humans; Injections, Intramuscular; Ischemia; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Neovascularization, Pathologic; Neovascularization, Physiologic; Peptide Fragments; Peptides; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Random Allocation; Recombinant Fusion Proteins; Recombinant Proteins; Sarcoma 180; Tumor Burden; Vascular Endothelial Growth Factor A

Expression of adrenomedullin, discovered as a vasodilatory peptide, is markedly up-regulated under pathological conditions such as tissue ischemia and inflammation, which are associated with neovascularization. Here, we tested the hypothesis that overly expressed adrenomedullin may augment collateral flow to ischemic tissues. We induced hindlimb ischemia in wild-type mice and injected a naked plasmid expressing human adrenomedullin or an empty vector into the ischemic muscle, followed by in vivo electroporation. Adrenomedullin markedly enhanced blood flow recovery as determined by Laser Doppler imaging. The mice treated with an empty vector suffered frequent autoamputation of the ischemic toe, which was completely prevented by adrenomedullin. Anti-CD31 immunostaining revealed that adrenomedullin significantly increased capillary density. The angiogenic effect of adrenomedullin was abrogated in endothelial nitric oxide synthase (eNOS)-deficient mice. These results indicate that adrenomedullin may promote collateral growth in response to ischemia through activation of eNOS.

Topics: Acute Disease; Adrenomedullin; Animals; Collateral Circulation; Female; Gene Expression; Gene Transfer Techniques; Humans; Ischemia; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Peptides; Recombinant Proteins; Vasodilation

Despite its safety and efficacy, the traumatic effects of high-energy shock waves (HESW) on renal morphology and function during long-term follow-up have yet to be elucidated. Although the main target of shock waves is the stone located in the kidney, the surrounding tissue and other organs are also subjected to trauma during this procedure. In contrast to renal blood flow evaluation after shock wave treatment, ischemic development, causing varying degrees of damage at the tissue level, has not been well evaluated. The renoprotective peptide adrenomedullin (AM) is a potent vasorelaxing, natriuretic and cell growth modulating peptide, which is thought to act as an autocrine/paracrine regulator in renal glomeruli and tubules. In this experimental study, renal parenchymal AM levels were assessed in an attempt to evaluate the effect of HESW on the tissue levels of this peptide, which may be responsible for the regulation of ischemia induced by extracorporeal shock wave lithotripsy(ESWL), in a rabbit model. Thirty white New Zealand rabbits, each weighing 3-5 kg were used. The animals were divided into three main groups, and varying numbers of shock waves (1000, 1500, 2000) were applied under fluoroscopic localization to the same kidney of all animals. Ketamine HCl anesthesia was administered (15-20 mg/kg) and all of the procedures were performed with a Multimed 2000 lithotriptor. Untreated contralateral kidneys were evaluated as controls. Following HESW application, the treated and untreated kidneys of each animal were removed through bilateral flank incisions under ketamine HCl anesthesia after 24 h and 7 days, respectively. Tissue AM levels were assessed with immunohistochemistry. During the early follow-up period (24 h), both treated and untreated kidneys showed a moderate to high degree of AM positivity. The number of tubules stained with AM increased as the number of shock waves increased and the expression of this protein became evident, possibly due to a higher degree of tissue damage. Additionally, a limited degree of AM positivity was noted in the contralateral kidneys although this was not as evident as the positivity seen in the treated kidneys. Assessment of tissue AM levels during late follow-up (7 days) in both kidneys demonstrated a moderate or limited degree of positivity in the treated kidneys. Limited or no positivity could be demonstrated in the contralateral kidneys at this time. Taking the certain traumatic effects of HESW, which causes tr

Topics: Adrenomedullin; Animals; Ischemia; Kidney; Kidney Diseases; Lithotripsy; Peptides; Rabbits; Renal Circulation

Adrenomedullin (ADM) is a vasodilator peptide, first isolated from human pheochromocytoma. To explore the pathophysiological role of ADM in ischemic conditions, we investigated the effects of hypoxia on ADM production and ADM mRNA expression in a cultured human colorectal carcinoma cell line, DLD-1. Northern blot analysis and radioimmunoassay showed that hypoxia stimulated the accumulation of ADM mRNA in the DLD-1 cells and immunoreactive ADM (ir-ADM) in the cultured media. Exposure to hypoxia for 12 hours increased ADM mRNA levels about 6-fold and ir-ADM levels about 4-fold. Moreover, treatment of DLD-1 cells with cobalt chloride, which mimics hypoxic states, significantly increased ADM mRNA levels about 18-fold and ir-ADM levels about 4-fold. These results suggest that ADM plays an important role in the pathophysiology of ischemic states.

Topics: Adrenomedullin; Cobalt; Colorectal Neoplasms; Culture Media; Gene Expression Regulation; Humans; Hypoxia; Ischemia; Peptides; RNA, Messenger; Tumor Cells, Cultured; Vasodilator Agents

The function of the vascular endothelium after storage at room temperature (24 degrees Celsius) for four, eight, and twenty-four hours was investigated with use of an ex vivo canine tibial perfusion model. Function was assessed in terms of changes in perfusion pressure and changes in the concentration of endothelin-1 in the venous effluent of the perfused tibiae. Endothelin-1 is a potent vasoconstrictor that is produced in low concentrations by normal endothelial cells and in increased concentrations by injured vascular endothelial cells. The mean perfusion pressures at flow rates of 1.0 and 1.5 milliliters per minute were significantly higher in the tibiae that had been stored for eight hours than in the tibiae that had been stored for four hours (p < 0.05), and they were significantly higher in the tibiae that had been stored for twenty-four hours than in the tibiae that had been stored for four or eight hours (p < 0.05). The increase in perfusion pressure with increasing duration of storage was associated with an increase in production of endothelin-1. The production of endothelin-1 in the tibiae that had been stored for eight hours (10.6 +/- 0.46 picograms per milliliter) was approximately ten times greater than that in the tibiae that had been stored for four hours (1.1 +/- 0.29 picograms per milliliter). The tibiae that had been stored for twenty-four hours had 19.1 +/- 1.5 picograms of endothelin-1 per milliliter, nearly twice that produced in the tibiae that had been stored for eight hours. Injection of acetylcholine demonstrated muscarinic receptor-mediated vasodilation in the tibiae that had been stored for four hours. In contrast, the tibiae that had been stored for eight and twenty-four hours had no evidence of acetylcholine-induced vasodilation of baseline perfusion vascular smooth-muscle tone. However, there was some preservation of endothelium-dependent vascular smooth-muscle relaxation in the tibiae that had been stored for eight and twenty-four hours, as norepinephrine-induced vascular smooth-muscle contraction was significantly greater in the presence of N(G)-monomethyl-L-arginine acetate (p < 0.05). Moreover, in the second phase of the study, a bolus injection of calcium ionophore A23187 in tibiae that had been stored for twenty-four hours relaxed vascular smooth muscle. Adrenomedullin, a novel peptide with known vasodilator properties, relaxed vascular smooth muscle in all three groups and also attenuated the pressor response to norepi

Topics: Acetylcholine; Adrenomedullin; Animals; Calcimycin; Dogs; Dose-Response Relationship, Drug; Endothelin-1; Endothelium, Vascular; Female; In Vitro Techniques; Ionophores; Ischemia; Male; Muscle, Smooth, Vascular; Nitric Oxide; Oligopeptides; Peptides; Pressoreceptors; Reperfusion Injury; Temperature; Tibia; Vasodilator Agents