Adrenomedullin Gene Research Articles

Mice heterozygous for adrenomedullin exhibit a more extreme inflammatory response to endotoxin-induced septic shock

Adrenomedullin (AM) is a highly conserved peptide that can act as a potent vasodilator, anti-microbial factor and anti-inflammatory factor. Several studies have implicated diverse roles for AM in regulating the inflammatory and hemodynamic responses to septic shock. Moreover, during sepsis the receptors that mediate AM signaling [calcitonin receptor-like receptor ( calcrl) and receptor activity modifying proteins ( RAMP) 2 and 3] undergo dynamic and robust changes in their expression. Although numerous studies have used animal models to study the role of administered or increased AM in septic animals, genetic studies to determine the consequences of reduced AM during septic shock have not yet been performed. Here, we used a murine model of lipopolysaccharide (LPS)-induced septic shock to assess the inflammatory response in mice heterozygous for the AM gene. Following LPS challenge, AM +/− mice had higher expression of TNF-α and IL-1β than LPS-treated wild-type (WT) controls. Consequently, serum TNF-α was also significantly elevated in LPS-treated AM +/− mice compared to WT LPS-treated mice. We also observed higher serum levels of liver enzymes, suggesting more advanced end-organ damage in mice with genetically reduced AM. Finally, we found that RAMP2 and calcrl expression levels were markedly reduced in LPS-treated mice, whereas RAMP3 expression was significantly elevated. Importantly, these changes in receptor gene expression were conserved in AM +/− mice, demonstrating that AM peptide itself does not impact directly on the expression of the genes encoding its receptors. We, therefore, conclude that during septic shock the dynamic modulation of AM and its receptors primarily functions to dampen the inflammatory response.

AMELIORATION OF CARDIAC FUNCTION IN CHRONIC MYOCARDIAL INFARCTED RATS FOLLOWING ADMINISTRATION OF VECTOR pcDNA3.1AM

1. The present study was designed to examine the cardiovascular effects of intravenously administered pcDNA3.1AM, a recombinant non-virus vector carrying a rat adrenomedullin (AM) gene translation fragment, in rats with chronic cardiac dysfunction induced by ligation of the left descending coronary artery. 2. Haemodynamic parameters were recorded by intraventricular catheterization. In situ hybridization and polymerase chain reaction (PCR) were performed to identify the distribution of the introduced vector. The concentration of AM was determined by radioimmunoassay. 3. Progressive cardiac dysfunction was observed following coronary artery ligation, as indicated by a significant reduction in mean arterial pressure (MAP) and increases in both central venous pressure (CVP) and end-diastolic pressure of the left ventricle (LVEDP; P < 0.01). Administration of pcDNA3.1AM significantly attenuated the progressive cardiac dysfunction and lowered the elevated CVP and LVEDP. The introduced vector was widely distributed in different organs, including the lungs, kidney, heart, liver, spleen and brain. However, intense staining of pcDNA3.1 AM was observed in the lungs and kidneys. The introduced vector was localized mainly in the endothelial cells of blood vessels. Radioimmunoassay showed elevated levels of AM in the plasma and lung and heart after surgery, but there was no significant further increase in the concentration of AM after pcDNA3.1AM delivery. 4. The present study has provided some novel findings on the potential beneficial effects of AM gene delivery on chronic cardiac function in rats. Expression of AM by a non-virus vector may also have therapeutic value against cardiac dysfunction in vivo.

Synergistic activation of the human adrenomedullin gene promoter by Sp1 and AP-2α

Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (−70/−29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2α (AP-2α), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2α synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (−103/−29) with Sp1 and AP-2α expression plasmids showed that Sp1 and AP-2α synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2α alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (−70/−29), whereas AP-2α bound to a more upstream promoter region (−103/−71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2α may be mediated by the binding of Sp1 to the promoter region (−70/−29) and the interaction with AP-2α, which binds to the promoter region (−103/−71).

Adrenomedullin and its receptors expression in renal tumors

10568 Background: Adrenomedullin (AM) is a 52 amino acid peptide that has an important role on tumor cell proliferation and neoangiogenesis through its receptors CRLR/RAMP2 and CRLR/RAMP3. AM gene expression is stimulated by Hypoxia Inductible Factor-1 (HIF- 1) and 60–80% of the human conventional renal carcinomas (cRCC) display mutations in the tumor suppressor protein von Hippel-Lindau, which leads to constitutively elevated HIF-1. Methods: Tumors and non-malignant kidney tissues were obtained from patients who underwent unilateral nephrectomy. We studied VEGFA, AM and its receptors expression by quantitative RT-PCR in 62 frozen renal tumors including: 40 cRCC; 5 cRCC metastasis; 5 chromophobe carcinomas; 5 Papillary carcinomas; 2 oncocytomas; 2 collecting duct carcinomas; 2 normal adjacent renal tissue; 1 unclassified renal tumor. AM and AM-receptors expression was studied in 42 paraffin-embedded renal tumors by immunohistochemistry using rabbit anti-AM, anti-CRLR, anti-RAMP2 and anti-RAMP3 polyclonal antibodies. Effects of these anti AM or anti AM- receptors antibodies on cell proliferation were examined in vitro on BIZ cell line (cRCC cells). Results: VEGFA, AM and AM-receptors genes were overexpressed in cRCC compared to other renal tumors (Except AM gene in chromophobe carcinoma). Their expressions were independent of classical prognostic factors (such as Fuhrman Grade and pT status). In cRCC, there was a strong positive correlation between VEGF-A gene expression and AM (r=0.7; p=0.01) and AM-receptors genes expressions (RAMP2 r=0.61; p= 0.01 and RAMP3 r=0.58; p= 0.01). Immunohistochemistry confirmed a tumor expression of CRLR, RAMP2 and AM in more than 80% cRCC. RAMP3 was expressed by inflammatory cells but not by tumoral cells. The cell proliferation assay showed a significant inhibition of BIZ cell proliferation by AM antibody or AM-receptors antibodies. Conclusion: AM and its receptor CRLR/RAMP2 are overexpressed in cRCC. In vitro cell proliferation assay results support that AM inhibition may suppress cRCC growth, independently of potential antiangiogenic effects. Further studies on animal models are needed but AM pathway may be a potential therapeutic target in cRCC. No significant financial relationships to disclose.

Adrenomedullin in the Kidney-Renal Physiological and Pathophysiological Roles

Adrenomedullin (AM) is a potent vasodilatory peptide originally discovered in human pheochromocytoma tissue. AM and AM gene expression are widely distributed in the cardiovascular system, including the kidney. The co-localization of AM and its receptor components such as calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP)2 and RAMP3 in the kidney, heart, and vasculature suggests an important role for the peptide as a regulator of renal, cardiac, and vascular function. Indeed, in addition to its cardiovascular effects, AM has renal vasodilatory, natriuretic, and diuretic actions. Consistent with these observations, immunohistochemical studies revealed that AM is stained in the collecting duct, distal convoluted tubules, vessels, and glomerular mesangial cells, endothelial cells and podocytes. Plasma AM levels are increased in patients with renal impairment in proportion to the severity of the disease. Previously we and other investigators showed that two molecular forms of AM, AM-glycine, an inactive form, and AM-mature, an active form, circulate in human plasma. Urine also contains both forms of AM; however, the AM-mature/AM-glycine ratio is higher in urine than in plasma. Interestingly, plasma AM-glycine and AM-mature levels are increased in renal failure, whereas urinary AM-glycine and AM-mature are decreased in this condition. These results indicate that the origin of urinary AM is different from that of plasma AM. Experimental studies showed that the renal tissue AM-mature/AM-glycine ratio is higher than that in plasma and urine. In addition, renal tissue concentrations of AM are increased in severely hypertensive rats. Considering that AM has antiapoptotic, antifibrotic, and antiproliferative effects, the increase of AM in renal disease may be a protective mechanism. In fact, AM gene delivery or long-term AM infusion significantly improved glomerular sclerosis, interstitial fibrosis, and renal arteriosclerosis in several malignant hypertensive models. This review describes the biochemistry, physiology, and circulating levels of AM and also discusses what is known about the pathophysiological role of AM in renal disease.

Adrenomedullin insufficiency increases allergen-induced airway hyperresponsiveness in mice

Adrenomedullin (ADM), a newly identified vasodilating peptide, is reported to be expressed in lungs and have a bronchodilating effect. We hypothesized whether ADM could be involved in the pathogenesis of bronchial asthma. We examined the role of ADM in airway responsiveness using heterozygous ADM-deficient mice (AM+/-) and their littermate control (AM+/+). Here, we show that airway responsiveness is enhanced in ADM mutant mice after sensitization and challenge with ovalbumin (OVA). The immunoreactive ADM level in the lung tissue after methacholine challenge was significantly greater in the wild-type mice than that in the mutant. However, the impairment of ADM gene function did not affect immunoglobulins (OVA-specific IgE and IgG1), T helper 1 and 2 cytokines, and leukotrenes. Thus the conventional mechanism of allergen-induced airway responsiveness is not relevant to this model. Furthermore, morphometric analysis revealed that eosinophilia and airway hypersecretion were similarly found in both the OVA-treated ADM mutant mice and the OVA-treated wild-type mice. On the other hand, the area of the airway smooth muscle layer of the OVA-treated mutant mice was significantly greater than that of the OVA-treated wild-type mice. These results suggest that ADM gene disruption may be associated with airway smooth muscle hyperplasia as well as enhanced airway hyperresponsiveness. ADM mutant mice might provide novel insights to study the pathophysiological role of ADM in vivo.

Differential regulation of adrenomedullin gene expression in the fundic and pyloric regions of the rat stomach during acute and chronic starvation

Adrenomedullin (AM) has been shown to be present in the stomach but the role of gastric AM is obscure. To investigate the effects of starvation on AM in the stomach, we studied the changes in gene expression of preproadrenomedullin (preproAM) and AM receptors by reverse transcription-polymerase chain reaction (RT-PCR), and tissue AM concentrations by radioimmunoassay (RIA) in the fundus and pylorus of the stomach of rats subjected to either acute (1-day) or chronic (4-day) starvation. An up-regulation of preproAM gene expression was observed in the fundus after acute starvation, and in the pylorus after chronic starvation. Immunoreactive AM (ir-AM) levels were increased in both fundus and pylorus after chronic starvation. In addition, marked reductions in the gene expression of fundic calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP) 3 as well as the pyloric CRLR and RAMP2 were observed in the chronically starved rats. The present study suggests that the gene expression of preproadrenomedullin mRNA is differentially regulated by starvation in the different parts of the stomach.

Adrenomedullin gene expression differences in mice do not affect blood pressure but modulate hypertension-induced pathology in males

Adrenomedullin (AM) is a potent vasodilator peptide in plasma at picomolar levels. Polymorphisms in the human AM gene have been associated with genetic predisposition to diabetic nephropathy and proteinuria with essential hypertension, and numerous studies have demonstrated that endogenous AM plays a role in protecting the heart and kidneys from fibrosis resulting from cardiovascular disease. Elevated plasma levels of AM are associated with pregnancy and sepsis and with cardiovascular stress and hypertension. However, there are no reports of the effects of genetic differences in the expression of the endogenous AM gene and of gender on blood pressure in these circumstances or on the pathological changes accompanying hypertension. To address these questions, we have generated mice having genetically controlled levels of AM mRNA ranging from approximately 50% to approximately 140% of wild-type levels. These modest changes in AM gene expression have no effect on basal blood pressure. Although pregnancy and sepsis increase plasma AM levels, genetically reducing AM production does not affect the transient hypotension that occurs during normal pregnancy or that is induced by treatment with lipopolysaccharide. Nor does the reduction of AM affect chronic hypertension caused by a renin transgene. However, 50% normal expression of AM enhances cardiac hypertrophy and renal damage in male, but not female, mice with a renin transgene. These observations suggest that the effect of gender on the role of AM in counteracting cardiovascular damage in humans merits careful evaluation.

Adrenomedullin gene expression differences in mice do not affect blood pressure but modulate hypertension-induced pathology in males

Adrenomedullin gene expression differences in mice do not affect blood pressure but modulate hypertension-induced pathology in males

Participation of adrenomedullin and its relation with vascular endothelial growth factor in androgen regulation of prostatic blood flow in vivo

Objectives We had previously reported that androgen-regulated prostatic blood flow and vascular endothelial growth factor (VEGF) were involved in the signal transduction pathway. Adrenomedullin (ADM) is a multifunctional regulatory peptide with mitogenic and angiogenic capabilities that are regulated by androgen. ADM is abundantly expressed in the prostate. We focused on ADM and evaluated its participation and relation with VEGF in androgen prostatic blood flow regulation using a castrated rat model. Methods We examined the effect of locally injected dihydrotestosterone (DHT) and ADM, and the co-administration of DHT with an ADM receptor antagonist (ADM 22-52) on prostatic blood flow. Furthermore, prostatic blood flow was evaluated after ADM and VEGF administration with each other’s antagonist, VEGF neutralizing antibody and ADM 22-52, respectively. Changes in the mRNA expression levels of ADM in the prostate after castration and successive androgen stimulation were also evaluated. Results The administration of ADM promptly increased prostatic blood flow in a dose-dependent manner within 30 minutes. The DHT-induced increase in prostatic blood flow was completely abolished by co-administration with anti-ADM. Anti-ADM inhibited the VEGF-induced prostatic blood flow elevation, but a VEGF neutralizing antibody did not affect the ADM-mediated blood flow elevation. Furthermore, upregulation of the ADM gene induced by DHT was inhibited by co-administration with a VEGF-neutralizing antibody. Conclusions These results have clearly demonstrated the direct regulation of prostatic blood flow by ADM and its involvement in androgenic prostatic blood flow regulation. Furthermore, ADM was estimated to be a downstream mediator of VEGF action in the signal transduction pathway.

Gene silencing of human RAMP2 mediated by short-interfering RNA

Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where the endogenous AM system has been suppressed. For this task we developed a technique to silence the AM gene in human umbilical vein endothelial cells (HUVECs) and the human embryonal kidney cell line (HEK-293). AM acts via two subtypes of receptor, named AM1 and AM2, which derive from the interaction of the calcitonin receptor-like receptors with two chaperones, called receptor activity modifying proteins (RAMP2 and RAMP3). Hence, we developed a protocol to suppress the human AM1 receptor by silencing the RAMP2 gene by transfection with short interfering RNAs (siRNAs). HUVECs were transfected using a new Ambion transfection reagent. RAMP2 gene silencing was determined in HUVECs by measuring RAMP2 mRNA levels in transfected and control cells by real-time polymerase chain reaction. The RAMP2 gene silencing was approximately 60% and was observed 48 h after transfection. Matrigel assay in vitro was carried out to evaluate the effects of siRNA sequences. HUVECs cells were plated on matrigel and the analysis of capillary-like tubule formation showed that the cells were viable. The knockdown of the RAMP2 gene decreased the formation of tubes in response to 10(-8) M AM. The conclusion is drawn that siRNA technology can be successfully used in the investigations on AM and AM receptor functions.

Adrenomedullin in adipocyte differentiation of human mesenchymal stem cells

Expression of adrenomedullin (AM), a potent vasodilator peptide, was studied during adipocyte differentiation of human mesenchymal stem cells (hMSCs). Immunoreactive AM levels in the medium were increased at day 4 and 8 of the adipocyte differentiation. Northern blot analysis showed increased expression of AM mRNA in hMSCs-derived adipocytes at day 4, 8, 12, and 18. Transient transfection assay showed that the promoter activity was higher in hMSCs-derived adipocytes than in hMSCs, when cells were transfected with plasmids containing a cis-acting region (−70/−29) of the human AM gene. Electrophoretic mobility shift assay showed that specific bands bound to the region (−70/−29) in hMSCs-derived adipocytes but not in hMSCs, and were abolished by the stimulatory protein 1 (Sp1) antibody. The present study has shown that AM expression is up-regulated during adipocyte differentiation of hMSCs probably via the interaction between Sp1 or Sp1-related factor(s) and the AM promoter region (−70/−29).

Reduced maternal expression of adrenomedullin disrupts fertility, placentation, and fetal growth in mice

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. Plasma AM expression dramatically increases during pregnancy, and alterations in its levels are associated with complications of pregnancy including fetal growth restriction (FGR) and preeclampsia. Using AM+/- female mice with genetically reduced AM expression, we demonstrate that fetal growth and placental development are seriously compromised by this modest decrease in expression. AM+/- female mice had reduced fertility characterized by FGR. The incidence of FGR was also influenced by the genotype of the embryo, since AM-/- embryos were more often affected than either AM+/- or AM+/+ embryos. We demonstrate that fetal trophoblast cells and the maternal uterine wall have coordinated and localized increases in AM gene expression at the time of implantation. Placentas from growth-restricted embryos showed defects in trophoblast cell invasion, similar to defects that underlie human preeclampsia and placenta accreta. Our data provide a genetic in vivo model to implicate both maternal and, to a lesser extent, embryonic levels of AM in the processes of implantation, placentation, and subsequent fetal growth. This study provides the first genetic evidence to our knowledge to suggest that a modest reduction in human AM expression during pregnancy may have an unfavorable impact on reproduction.

Blood pressure and urinary sodium excretion in relation to the A-1984G adrenomedullin polymorphism in a Chinese population

Adrenomedullin (ADM) is a vasodilator and inhibits salt appetite. An A-to-G substitution at position -1984 in the promoter region of the ADM gene likely increases transcription. We therefore investigated this polymorphism in relation to blood pressure and urinary sodium in a Chinese population. We genotyped 427 Chinese enrolled in a family-based population study. We measured blood pressure by conventional sphygmomanometry and ambulatory monitoring. The frequencies of the ADM AA, AG, and GG genotypes were 50.6, 38.2, and 11.2%, respectively. In adjusted analyses, G allele carriers, compared to AA homozygotes, had significantly lower conventional (45.3 versus 48.5 mm Hg, P = 0.004) and 24-h (42.6 versus 44.3 mm Hg, P = 0.03) pulse pressures and urinary sodium excretion (143.8 versus 159.4 mmol/day, P = 0.03). In parents, but not offspring, both systolic pressure and pulse pressure were significantly (P<0.01) lower in G allele carriers. The genotypic difference in sodium excretion was consistent across the age range. In 68 informative offspring, transmission of the G allele was associated with lower urinary sodium excretion (effect size, 40.1 mmol/day, P = 0.01). In 81 healthy volunteers, the plasma ADM concentration was 15.2% higher in GG homozygotes than in sex- and age-matched AA subjects (11.4 versus 9.9 pmol/l, P = 0.10). In conclusion, in Chinese, the ADM -1984G allele is associated with lower sodium excretion and in older subjects also with lower systolic pressure and narrower pulse pressure.

The estrogen-responsive adrenomedullin and receptor-modifying protein 3 gene identified by DNA microarray analysis are directly regulated by estrogen receptor

Recent studies have revealed that hundreds of genes in the uterus are activated by estrogen. Their expression profiles differ over time and doses and it is not clear whether all these genes are directly regulated by estrogen via the estrogen receptor. To select the genes that may be regulated by estrogen, we treated mice with several doses of estrogen and searched for those genes whose dose-response expression pattern mirrored the uterine growth pattern. Among those genes, we found that the dose-dependent expression of the adrenomedullin (ADM) gene correlated well with the uterotrophic effect of estrogen. ADM expression is induced early after estrogen administration and is restricted to the endometrial stroma. The spatiotemporal gene expression pattern of ADM was similar to that of receptor-modifying protein 3 (RAMP3). RAMP3 is known to modify calcitonin gene-related receptor (CRLR) so that it can then serve as an ADM receptor. Chromatin immunoprecipitation assays indicated that the estrogen receptor binds directly to the ADM promoter region and RAMP3 intron after estrogen administration. It was also shown that neither the ADM nor RAMP3 gene could be activated in estrogen receptor-alpha-null mouse. Although uterine ADM expression has been reported to occur in the myometrium, our observations indicate that estrogen-induced ADM is also expressed in the uterine stroma and that such variable, spatiotemporally regulated ADM expression contributes to a wider range of biological effects than previously expected.

Identification of adipocyte differentiation-related regulatory element for adrenomedullin gene repression (ADRE-AR) in 3T3-L1 cells

Adrenomedullin (AM), a potent vasodilator peptide, has been suggested to act against cardiovascular complications and insulin resistance in the metabolic syndrome. We have already reported the AM gene repression in the early phase of adipocyte differentiation of NIH 3T3-L1 cells. Here we show adipocyte differentiation-related regulatory element for AM gene repression (ADRE-AR) in 36-bp region (−2135/−2100) of the AM gene. 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. On the third day of differentiation, the promoter function was analyzed using the reporter plasmids, which contain the promoter region of AM gene (−4616/+108) in pGL3-basic luciferase reporter vector. The promoter activity decreased to about 20% in 3T3-L1 adipocytes when compared with 3T3-L1 preadipocytes, and a 36-bp region (−2135 to −2100) upstream from the transcription initiation site of the AM gene was necessary for higher AM gene expression in preadipocytes. This 36-bp ADRE-AR contains three copies of G/AAAA sequence (5′- GAAAT GAAAGT AAAA-3′) (−2124/−2110), which are conserved between mouse and human, and the introduction of mutations in each copy of G/AAAA sequence decreased the promoter activity in preadipocytes and adipocytes. Electrophoretic mobility shift assay showed that the full-length ADRE-AR was specifically bound by a certain nuclear protein(s). The present study has raised the possibility that ADRE-AR may play important roles in the AM gene expression in preadipocytes, and that the AM gene may be repressed through the ADRE-AR in adipocytes.

Adrenomedullin gene transfer induces neointimal apoptosis and inhibits neointimal hyperplasia in injured rat artery

Arterial wall injury leads to inflammatory reaction and release of growth factors that may mediate intimal regrowth. It is hypothesized that the neointimal cells may originate from adventitial myofibroblasts, medial smooth muscle cells, or differentiated bone marrow derived cells. Adrenomedullin (AM), an auto/paracrine cardiovascular peptide that is secreted from fibroblasts, endothelial cells, and vascular smooth muscle cells, may have a regulatory role in the intimal regeneration. In order to investigate the role of AM in neointimal growth, stimulation of stem cell migration, and apoptosis, we overexpressed AM with recombinant adenovirus in a rat arterial injury model. The intimae were significantly thinner in the arteries treated with AM adenovirus compared to the control group. Intima/media ratios were 0.48 +/- 0.18 and 1.01 +/- 0.20 (P < 0.05) in the AM group and the control group, respectively. In addition, a significantly higher apoptotic index of neointimal cells was seen in the AM gene transfer group compared to the control (2.78 +/- 0.5 vs. 0.57 +/- 0.20, P < 0.01). The neointimal cells stained positive for alpha-smooth muscle actin and negative for desmin suggesting possible myofibroblast origin. Very few c-Kit+ or MDR1+ cells were detected 2 weeks after the injury. We conclude that AM overexpression inhibits neointimal growth. The inhibition is associated with enhanced apoptosis of the neointimal cells which may be of myofibroblast origin.

Hypoxic stress-enhanced expression and release of adrenomedullin (AM) and up-regulated AM receptors, while glucose starvation reduced AM expression and release and down-regulated AM receptors in monkey renal cells

The proliferative peptide adrenomedullin (AM) has a wide distribution in a variety of tissues and cells. The mechanism how the AM gene is regulated in cells is not yet known. The renal cortex, renal vascular smooth muscles, glomeruli and tubular epithelial cells are very sensitive to hypoxia. Renal hypoxia produces acute renal tubular necrosis and markedly induces AM expression in damaged cells. However, little information is available regarding the possible pathophysiological production and release of renal tubular AM. Regulation of membrane-bound AM receptors in renal cells has not yet been systematically studied. To elucidate the potential pathological role of human AM we examined the production and release of AM, as well as the characteristics of surface membrane AM receptors in cultured monkey renal tubular epithelial cells (RC) exposed to hypoxia, induced with endothelin-1, and subjected to glucose deprivation. Exposure of RC to hypoxia (1 % O(2), 5 % CO(2) in N(2)), and to phorbol 12-myristate 13-acetate (PMA) increased production and secretion of AM and increased specific [(125)I]AM binding on RC. Metabolic stress (1 % glucose in the cultivation medium) and preincubation of RC with rival peptide endothelin-1 significantly reduced immunoreactive-AM in a conditioned medium and whole cell surface membrane AM binding on RC. Altogether, our data suggest that the AM is involved in the adaptation of renal tubular cells to hypoxia. Increased expression of AM mRNA and regulation of AM receptors in metabolic stress may function as an important autocrine/paracrine regulator(s) of renal tubular epithelial cells.

Postischemic infusion of adrenomedullin protects against ischemic stroke by inhibiting apoptosis and promoting angiogenesis

Adrenomedullin (AM) is a peptide hormone widely distributed in the central nervous system. Our previous study showed that AM gene delivery immediately after middle cerebral artery occlusion (MCAO) protected against cerebral ischemia/reperfusion (I/R) injury by promoting glial cell survival and migration. In the present study, we investigated the effect of delayed AM peptide infusion on ischemic brain injury at 24 h after MCAO. AM infusion significantly reduced neurological deficit scores at days 2, 4, and 8 after cerebral I/R. AM reduced cerebral infarct size at 8 and 15 days after surgery as determined by quantitative analysis. Double staining showed that AM infusion reduced TUNEL-positive apoptotic cells in both neurons and glial cells, as well as reduced caspase-3 activity in the ischemic area of the brain. In addition, AM treatment increased capillary density in the ischemic region at 15 days after I/R injury. Parallel studies revealed that AM treatment enhanced the proliferation of cultured endothelial cells as measured by both 3H-thymidine incorporation and in situ BrdU labeling. Both in vitro and in vivo AM effects were blocked by calcitonin gene-related peptide (8–37), an AM receptor antagonist. Moreover, AM’s effects were associated with increased cerebral nitric oxide (NO) levels, as well as decreased NAD(P)H oxidase activities and superoxide anion production. These results indicate that a continuous supply of exogenous AM peptide protects against I/R injury by improving the survival of neuronal and glial cells, and promoting angiogenesis through elevated NO formation and suppression of oxidative stress.

Potential of Gene Therapy Strategy for the Treatment of Hypertension

Hypertension is one of the most important risk factors for cardiovascular diseases (CVDs). CVD extends across all populations and represents the leading cause of morbidity and mortality in industrialized countries. Hypertension increases the risk of peripheral arterial disease, cardiomyopathy, stroke, and renal failure. The disease affects &50 million Americans and goes undetected in at least one third of the population. In spite of the tremendous progress made by pharmacotherapy in the management and control of hypertension, there has been a steady escalation in its prevalence during the last decade. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau and that novel and innovative approaches must be sought for the treatment, control, and possible cure of hypertension. As a result, efforts of our group and those of many others have been diverted to explore the use of gene transfer and gene therapy strategies for a long-term control of hypertension. We have argued that gene therapy offers potentially significant improvements and benefits over the use of traditional pharmacotherapy: (1) noncompliance by patients can be significantly reduced or even eliminated because of the fact that a single treatment involving gene transfer could remain effective for months or even years; (2) side effects associated with pharmacotherapy can be minimized as a result of specific targeting of a therapeutic gene in a given tissue and optimally influencing cardiovascular pathophysiology on an individual patient basis; and (3) with its potential to produce long-term beneficial outcomes in end-organ damage, the gene therapy strategy could lead to a cure of hypertension and its related pathophysiology. It is noteworthy to mention that current pharmacotherapies are often unable to reverse end-organ damage associated with hypertension. In order for the gene therapy for hypertension to be successful, one must provide conceptual support of its efficacy in …