Potent antiproliferative activity of bradykinin B2 receptor selective agonist FR-190997 and analogue structures thereof: A paradox resolved?

Βradykinin stimulation of B2 receptor is known to activate the oncogenic ERK pathway and overexpression of bradykinin receptors B1 and B2 has been reported to occur in glioma, colorectal and cervical cancers. B1R and B2R antagonists have been shown to reverse tumor proliferation and invasion. Paradoxically, B1R and B2R agonism has also been reported to elicit antiproliferative benefits. In order to complement the data accumulated to date with the natural substrate bradykinin and peptidic B2R antagonists, we decided to examine for the first time the response elicited by B2R stimulation in breast cancer lines with a non-peptidic small molecule B2R agonist. We synthesized and assessed the highly selective and potent B2R partial agonist FR-190997 in MCF-7 and MDA-MBA-231 breast cancer lines and found it possessed significant antiproliferative activity (IC50 2.14 and 0.08 µΜ, respectively). The modular nature of FR-190997 allowed us to conduct a focused SAR study and discover compound 10 which exhibits subnanomolar antiproliferative activity (IC 50 0.06 nΜ) in the TNBC MDA-MBA-231 cell line. This performance surpasses, in most cases by several orders of magnitude, those of established anticancer agents and FDA-approved breast cancer drugs. In line with the established literature we suggest that this remarkable activity precipitates from a dual mode of action involving agonist-induced receptor internalization/degradation combined with sequestration of functional intracellular B2 receptors and inhibition of the associated endosomal signaling. The latter mode may be realized by appropriate ligands regardless of B2R agonist/antagonist designation which only relates to membrane residing GCPRs. Under this prism the controversy over the antiproliferative effects of B2 agonists and antagonists is potentially neutralized.

Genes and genetic variations involved in the development of hypertension: focusing on a Greek patient cohort.

INTRODUCTION: Essential hypertension (HTN) is a multifactorial disease involving environmental, genetic and other factors. Over the past years, genetic studies of essential HTN have increased dramatically but the molecular mechanisms involved are still unknown. As part of a research program coordinated by Boston university (USA), we studied the role of various genes and single nucleotide polymorphisms (SNPs) in the inheritance or the onset of HTN in African-American, Caucasian-American and Greek families. METHODS: Among 128 Greek families with a history of HTN, we studied 1474 people. Of the total examined, 273 men and 286 women were hypertensive. Based on 410 DNA samples from the hypertensive subjects, different SNPs were examined. An overall meta-analysis of the results from the Greek families, as well as a comparison with the 2 other groups (African-Americans and Caucasian-Americans), was performed. RESULTS: We report SNPs that are associated with the inheritance of HTN and are located either at the promoters of N-methyltransferase and catalase genes, or within the coding region of NEDD4L ubiquitin ligase gene, or SNPs in mitochondrial DNA of hypertensive probands. Furthermore, we clarified the role of hereditary predisposition in the development of HTN, showing that the presence of maternal HTN was significantly higher in African-Americans and Greeks compared to Caucasian-Americans (81.7%, 84.8%, and 65%), while the paternal HTN showed no such difference (50%, 48.3% and 44.9%), respectively. CONCLUSIONS: Although genetic factors that were correlated with HTN were identified, it was not possible to identify a single gene that should be targeted for the treatment of HTN. Nevertheless, the important role of the maternal hereditary predisposition to HTN in the Greek patients and the responsible genetic factors involved should be further examined.

Blockade of platelet alpha2B-adrenergic receptors: a novel antiaggregant mechanism.

BACKGROUND: Platelets play a vital role in hemostasis and thrombosis. Catecholamines have a profound effect on platelet aggregation and atherothrombosis but the exact mechanism involved is insufficiently understood. In this report, we demonstrate the existence and role of alpha2B-adrenergic receptors (α2B-ARs) in normal human platelets. METHODS: Sixteen healthy individuals were recruited as donors of normal blood from which platelets were isolated. The presence of α2B-ARs in platelets was proven by Western blot analysis. In order to investigate their function, we performed light transmittance aggregometry and platelet function activity tests by examining the inhibitory effects of specific α2B-AR antibodies and of the selective α2B-AR antagonist ARC 239. RESULTS: Pretreatment of human platelets with agents that selectively block α2B-ARs showed a substantial inhibition in platelet aggregation that had been induced by adenosine diphosphate (ADP), by epinephrine and by arachidonic acid. The percent aggregation decreased from 81.5 ± 1.7% to 35.8 ± 5% and to 24 ± 4.6% for ADP with α2B-Abs and ARC 239 respectively, from 72.2 ± 1.9% to 25.5 ± 4.3% and to 8.8 ± 1.7% for epinephrine with α2B-Abs and ARC 239 respectively, and from 87 ± 2.1% to 47.9 ± 6.2% and to 61.2 ± 5.7% for arachidonic acid with α2B-Abs and ARC 239 respectively, p<0.05 for all. Additionally, collagen/epinephrine closure time increased from 120.8 ± 6.1s to 189.5 ± 39.5s (p=0.001). CONCLUSIONS: Our results reveal that contrary to previous knowledge, the α2B-AR subtype does exist in platelets and is an important regulator of aggregation. Inhibition of α2B-ARs in platelets may offer a novel therapeutic opportunity in the prevention of atherothrombotic events.

'Volume-expanded' hypertension: the effect of fluid overload and the role of the sympathetic nervous system in salt-dependent hypertension.

It is widely believed that salt-dependent hypertension is induced and maintained by expansion of intravascular fluid volume resulting from excessive retention of sodium. The purpose of this brief article is to present a series of arguments in support of the thesis that volume overload per se does not raise the arterial blood pressure. Several investigators in the 1960s and 1970s reported that excessive retention of salt - regardless of cause - leads to sympathetic activation mediated by the effects of the Na ion on α(2)-adrenergic receptors located mostly in the brainstem. In recent years, the cloning and characterization of α(2)-adrenergic receptors subtypes permitted differentiation of their hemodynamic effects via use of salt loading of nephrectomized animals submitted to genetic engineering or gene treatment. These studies indicate that sodium alters the balance between the sympathoinhibitory α(2A)-adrenergic receptors and the sympathoexcitatory α(2B)-adrenergic receptors, leading to a hyperadrenergic hypertensive state unrelated to volume overload.

A2b adenosine receptor regulates hyperlipidemia and atherosclerosis.

BACKGROUND: The cAMP-elevating A(2b) adenosine receptor (A(2b)AR) controls inflammation via its expression in bone marrow cells. METHODS AND RESULTS: Atherosclerosis induced by a high-fat diet in apolipoprotein E-deficient mice was more pronounced in the absence of the A(2b)AR. Bone marrow transplantation experiments indicated that A(2b)AR bone marrow cell signals alone were not sufficient to elicit this effect. Intriguingly, liver expression of the A(2b)AR in wild-type mice was vastly augmented by a high-fat diet, raising the possibility that this upregulation is of functional significance. A(2b)AR genetic ablation led to elevated levels of liver and plasma cholesterol and triglycerides and to fatty liver pathology typical of steatosis, assessed by enzymatic assays and analysis of liver sections. Western blotting and quantitative polymerase chain reaction revealed elevated expression of the following molecules in the liver of A(2b)AR-null mice: the transcription factor sterol regulatory element binding protein-1 (SREBP-1) and its 2 downstream targets and regulators of lipogenesis, acetyl CoA carboxylase and fatty acid synthase. Pharmacological activation or inhibition of A(2b)AR in primary hepatocytes confirmed the regulation of SREBP-1 by this receptor. A(2b)AR-mediated changes in cAMP were found to regulate levels of the transcriptionally active form of SREBP-1. Finally, adenovirally mediated restoration of the A(2b)AR in the liver of A(2b)AR-null mice reduced the lipid profile and atherosclerosis. Similarly, in vivo administration of the A(2b)AR ligand BAY 60-6853 in control mice on a high-fat diet reduced the lipid profile and atherosclerosis. CONCLUSION: This study provides the first evidence that the A(2b)AR regulates liver SREBP-1, hyperlipidemia, and atherosclerosis, suggesting that this receptor may be an effective therapeutic target.

Cardioprotective properties of bradykinin: role of the B(2) receptor.

Following the introduction of angiotensin-converting enzyme (ACE) inhibitors in the treatment of hypertension and ischemic heart disease, there has been increasing interest in the bradykinin-mediated aspects of ACE inhibition. Several preclinical and clinical studies have been conducted using genetically engineered animals or pharmacological agonists and antagonists of the two receptors of bradykinin, B(1)R and B(2)R. The results have mostly indicated that the B(1)R, whose expression is induced by tissue damage, seem to have mostly noxious effects, whereas the constitutively expressed B(2)R, when activated, exert mostly beneficial actions. Accumulating evidence in the recent literature suggests that the B(2)R have an important role in the process of ischemic post-conditioning that limits the ischemia/reperfusion injury of the myocardium. In this article, we describe a series of experiments conducted on mice submitted to acute myocardial infarct and treated either with ACE inhibition (which produces potentiation of bradykinin resulting in non-selective B(1)R and B(2)R activation) or with a potent and highly selective B(2)R agonist. These data suggest that this latter pharmacological approach offers functional and structural benefits and is therefore a promising cardioprotective therapeutic modality against acute ischemic events.

Cardioprotective effects of a selective B(2) receptor agonist of bradykinin post-acute myocardial infarct.

BACKGROUND: The cardioprotective benefits of bradykinin are attributable to activation of its B(2) receptor (B(2)R)-mediated actions and abolished by B(2)R antagonists. The current experiments evaluated the cardioprotective potential of a potent, long-acting B(2)R-selective agonist peptide analogue of bradykinin, the compound NG291. METHODS: We compared the extent of cardiac tissue damage and remodeling and expression pattern of selected genes in mice submitted to acute myocardial infarct (MI) and treated for 1 week with either NG291 [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin or with saline delivered via osmotic minipump. RESULTS: Active treatment resulted in better ejection fraction (EF) 69 +/- 1% vs. 61 +/- 3.1% (P = 0.01), (vs. 85 +/- 1.3% in sham-operated controls), fractional shortening (FS) 38 +/- 4% vs. 32 +/- 8% (NS) (vs. 53 +/- 1.2 in sham-operated controls), and fewer markers of myocyte apoptosis (TUNEL-positive nuclei 4.9 +/- 1.1% vs. 9.7 +/- 0.03%, P = 0.03). Systolic blood pressure (SBP) at end point was normal at 110 +/- 4.2 in actively treated mice, but tended to be lower at 104 +/- 4.7 mm Hg in saline controls with decreased cardiac systolic capacity. Expression patterns of selected genes to factors related to tissue injury, inflammation, and metabolism (i.e., the B(1)R, B(2)R, endothelial nitric oxide synthase (eNOS), TNF-alpha, cardiomyopathy-associated 3 (Cmya3), and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4)) showed that acute MI induced significant upregulation of these genes, and active treatment prevented or attenuated this upregulation, whereas the B(2)R agonist itself produced no difference in the myocardium of sham-operated mice. CONCLUSIONS: Treatment with a selective B(2)R agonist initiated at the time of induction of acute MI in mice had a beneficial effect on cardiac function, tissue remodeling, and inflammation-related tissue gene expression, which may explain its structural and functional benefits.

Modulation of angiotensin II-mediated cardiac remodeling by the MEF2A target gene Xirp2.

RATIONALE: The vasoactive peptide angiotensin II (Ang II) is a potent cardiotoxic hormone whose actions have been well studied, yet questions remain pertaining to the downstream factors that mediate its effects in cardiomyocytes. OBJECTIVE: The in vivo role of the myocyte enhancer factor (MEF)2A target gene Xirp2 in Ang II-mediated cardiac remodeling was investigated. METHODS AND RESULTS: Here we demonstrate that the MEF2A target gene Xirp2 (also known as cardiomyopathy associated gene 3 [CMYA3]) is an important effector of the Ang II signaling pathway in the heart. Xirp2 belongs to the evolutionarily conserved, muscle-specific, actin-binding Xin gene family and is significantly induced in the heart in response to systemic administration of Ang II. Initially, we characterized the Xirp2 promoter and demonstrate that Ang II activates Xirp2 expression by stimulating MEF2A transcriptional activity. To further characterize the role of Xirp2 downstream of Ang II signaling we generated mice harboring a hypomorphic allele of the Xirp2 gene that resulted in a marked reduction in its expression in the heart. In the absence of Ang II, adult Xirp2 hypomorphic mice displayed cardiac hypertrophy and increased beta myosin heavy chain expression. Strikingly, Xirp2 hypomorphic mice chronically infused with Ang II exhibited altered pathological cardiac remodeling including an attenuated hypertrophic response, as well as diminished fibrosis and apoptosis. CONCLUSIONS: These findings reveal a novel MEF2A-Xirp2 pathway that functions downstream of Ang II signaling to modulate its pathological effects in the heart.

Cell signaling, internalization, and nuclear localization of the angiotensin converting enzyme in smooth muscle and endothelial cells.

The angiotensin converting enzyme (ACE) catalyzes the extracellular formation of angiotensin II, and degradation of bradykinin, thus regulating blood pressure and renal handling of electrolytes. We have previously shown that exogenously added ACE elicited transcriptional regulation independent of its enzymatic activity. Because transcriptional regulation generates from protein-DNA interactions within the cell nucleus we have investigated the initial cellular response to exogenous ACE and the putative internalization of the enzyme in smooth muscle cells (SMC) and endothelial cells (EC). The following phenomena were observed when ACE was added to cells in culture: 1) it bound to SMC and EC with high affinity (K(d) = 361.5 +/- 60.5 pM) and with a low binding occupancy (B(max) = 335.0 +/- 14.0 molecules/cell); 2) it triggered cellular signaling resulting in late activation of focal adhesion kinase and SHP2; 3) it modulated platelet-derived growth factor receptor-beta signaling; 4) it was endocytosed by SMC and EC; and 5) it transited through the early endosome, partially occupied the late endosome and the lysosome, and was localized to the nuclei. The incorporation of ACE or a fragment of it into the nuclei reached saturation at 120 min, and was preceded by a lag time of 40 min. Internalized ACE was partially cleaved into small fragments. These results revealed that extracellular ACE modulated cell signaling properties, and that SMC and EC have a pathway for delivery of extracellular ACE to the nucleus, most likely involving cell surface receptor(s) and requiring transit through late endosome/lysosome compartments.

Attenuation of angiotensin II-induced hypertension and cardiac hypertrophy in transgenic mice overexpressing a type 1 receptor mutant.

BACKGROUND: The angiotensin II (AngII) type 1 receptor (AT1) regulates cardiovascular function by activating various signal pathways. The purpose of this study was to evaluate the effects of a mutant AT1 receptor on AngII-responding blood pressure and cardiac hypertrophy in conjunction with altered AngII activation of RhoA and Akt. METHODS: A mutant AT1 receptor was constructed and overexpressed in C57BL mice using a ubiquitous-expression vector pCAGGS. The phenotype and signal transduction of the transgenic (TG) mice were compared with the wild-type (WT) mice. RESULTS: The TG mice showed a similar baseline phenotype as WT mice, but their blood pressure in response to continuous AngII infusion was significantly lower, as measured on days 3, 4, 7, and 14, with a difference of 20 mm Hg by day 14. There was also a significantly larger heart-to-total-body-weight ratio in the WT mice, whose heart weight (HW) was 0.441 +/- 0.008% of total body weight (BW) compared to the TG mice at 0.416 +/- 0.008%. Aortic endothelial cells isolated from these TG mice displayed an altered signaling profile, such as diminished activation of Akt and RhoA in response to AngII. In contrast, Galphaq coupling and ERK/JNK activation did not change. CONCLUSION: The expression of an AT1 mutant receptor in the presence of WT receptor can effectively modulate AngII-effected signaling. Furthermore, the elimination of Akt and RhoA activation by AngII significantly reduces but does not eliminate its hypertensive effect.

Hypertension in transgenic mice with brain-selective overexpression of the alpha(2B)-adrenoceptor.

BACKGROUND: Previous studies have shown that the presynaptic alpha(2B)-adrenoceptor subtype in the central nervous system has a sympathoexcitatory function and its activation leads to a hyperadrenergic hypertensive state. The purpose of this project was to develop a novel hyperadrenergic model, a transgenic (TG) mouse model with brain-selective overexpression of the alpha(2B)-adrenergic receptor (alpha(2B)-AR). METHODS: We used Southern blot analysis to confirm transgene, real-time PCR to assess gene expression, western Blot analysis and immunohistology to assess protein expression and localization in brain areas. Indirect blood pressure (BP) and heart rate were recorded. RESULTS: In TG mice there was a 1.8-fold increase in alpha(2B)-AR protein expression compared to wild-type (WT) mice. Immunostaining of brain sections revealed that concentration of alpha(2B)-AR was much more pronounced in TG than in WT mice. Systolic BP at 8 weeks of age was significantly elevated in TG 130 +/- 6 mm Hg, compared with WT control nontransgenic littermates of the same age 107 +/- 7 mm Hg, (P < 0.05), indicating that the TG mice had indeed developed hypertension. CONCLUSIONS: We have therefore documented that overexpression of the alpha(2B)-AR gene leads to increased production of alpha(2B)-AR protein in brain regions known to regulate central sympathetic outflow, thus resulting in sustained BP elevation. This is a unique model of experimental hypertension driven purely by overexpression of the alpha(2B)-AR that would result in an overactive sympathetic system and would be suitable for testing the pharmacologic properties of potential therapeutic agents.

Pleiotropic effects of statins may improve outcomes in atherosclerotic renovascular disease.

BACKGROUND: Atherosclerotic renovascular disease (ARD) coexists with arterial obstructive disease in the coronary, cerebral, and peripheral arteries that may remain underdiagnosed and untreated. METHODS: This retrospective study compares overall survival and renal survival (i.e., time to doubling of serum creatinine or end-stage renal disease (ESRD)) over an 11-year period in 104 ARD patients of whom 68 received statin therapy (group S) because of elevated lipid levels and 36 had no statin (group NS) because of normal lipid profile at entry. RESULTS: Atherosclerosis in another vascular bed was documented in 84%. Lipid profiles at end point were virtually identical in both the groups. Group S had mean survival 123months (confidence interval (CI) 113-134) with four deaths, and mean renal survival 122months (CI 113-131). Group NS had mean survival 33 months (CI 23-42) with 13 deaths, and mean renal survival 27 months (CI 17-37). CONCLUSIONS: Statin therapy was associated with lesser rate of progression of renal insufficiency (with 7.4% of S patients reaching renal end points vs. 38.9% of NS patients) and lower overall mortality (5.9 % in S vs. 36.1% in NS patients), P < 0.001 for both. Although both groups received what was deemed optimal therapy, they did have other differences that may have affected the outcomes (a limitation addressed by Cox multiple regression analysis). These results suggest the need for prospective randomized controlled studies in ARD patients in order to explore potential benefits of statins that may not be attributable solely to lipid lowering.

Angiotensin-converting enzyme inhibition after experimental myocardial infarct: role of the kinin B1 and B2 receptors.

We sought to define the contribution of each of the 2 kinin receptors (bradykinin 1 receptor [B(1)R] and bradykinin 2 receptor [B(2)R]) to the cardioprotection of angiotensin-converting enzyme (ACE) inhibition after acute myocardial infarct. Wild-type mice and gene knockout mice missing either B(1)R or B(2)R were submitted to coronary ligation with or without concurrent ACE inhibition and had evaluation of left ventricular systolic capacity by assessment of fractional shortening (FS). Baseline FS was similar in all of the animals and remained unchanged in sham-operated ones. At 3 weeks after myocardial infarct, in the wild-type group there was a 27% reduction of FS (P<0.5) without ACE inhibition and 8% with ACE inhibition; in the B(1)R(-/-) groups the FS was reduced by 24% and was no different (at 28%) with ACE inhibition; in the B(2)R(-/-) groups, however, the FS was decreased by 39% and with ACE inhibition was decreased further by 52%. Analysis of bradykinin receptor gene expression in hearts showed that when one receptor was missing, the other became significantly upregulated; but the B(1)R remained highly overexpressed in the B(2)R(-/-) mice throughout, whereas the overexpressed B(2)R became significantly suppressed in the B(1)R(-/-) mice in a manner quantitatively and directionally similar to that of wild-type mice. We conclude that both bradykinin receptors contribute to the cardioprotective bradykinin-mediated effect of ACE inhibition, not only the B(2)R as believed previously; but, whereas with potentiated bradykinin in the absence of B(1)R, the upregulation of B(2)R is simply insufficient to provide full cardioprotection, in the absence of B(2)R, the upregulated B(1)R actually seems to inflict further tissue damage.

Inhibition of the alpha(1D)-adrenergic receptor gene by RNA interference (RNAi) in rat vascular smooth muscle cells and its effects on other adrenergic receptors.

Sympathetic-induced vasoconstriction is mediated by various adrenergic receptor (AR) subtypes located on membranes of vascular smooth muscle cells (VSMC) located on the arterial wall, but is mostly attributed to activation of the alpha(1D)-AR. In order to study interaction and cross-talk among AR genes, we induced post-transcriptional silencing of the alpha(1D)-AR gene in cultured VSMC using the RNAi technique. A pSEC neo expression plasmid vector containing a small interfering RNA (siRNA) sequence selected to bind to the targeted mRNA of the alpha(1D)-AR gene was transfected into cultured VSMC from rat aorta. The RNA expression of all AR-subtype genes was assessed by Q-RT-PCR and the alpha(1D) and alpha(2A)-AR proteins quantified by Western blot. In siRNA-transfected cells, the alpha(1D)-AR protein levels decreased by 55%, 69% and 75% at 24 h, 48 h and 72 h, respectively (p<0.03-0.01) with progressive increases in its gene expression by 50%-61% and concurrent increase in alpha(2A)-AR protein peaking at 48 h. Decreases were noted in expression of the alpha(1A), alpha(2A), and beta(3) AR genes. We conclude that post-transcriptional silencing of the alpha(1D)-AR gene leads to significant decrease in receptor protein despite reactive increase in gene expression. However, suppression of one AR leads to reactive changes in other subtypes, indicating that cross-talk among related genes, whose products have overlapping functions, may partly offset anticipated effects in vivo.

Frequency of coronary artery disease in patients with renal artery stenosis without clinical manifestations of coronary insufficiency.

Atherosclerotic renal artery stenosis (RAS) and coronary artery disease (CAD) arise from the same multiple risk factors. The purpose of this study was to assess the frequency of previously undiagnosed CAD in patients with angiographically confirmed RAS, by conducting coronary arteriography in the same setting. Of 57 consecutive patients referred for renal arteriography on clinical grounds during a 14-month period, 28 had no RAS and 6 had RAS, but previously documented CAD. Of the remainder 23 patients, 17 (74%; CI 56%-92%) had both RAS and CAD (7 single vessel, 4 two-vessel, and 7 multivessel disease). The clinical characteristics, such as age, blood pressure (BP) levels, signs of heart failure, were no different between those with and without CAD, although the 4 diabetic patients, the 4 patients with fundoscopic findings of grade III retinopathy, 11 of 14 with peripheral arterial disease, and 7 of 8 patients with prior stroke belonged in the CAD group. None developed complications as a result of the two consecutive procedures. The data suggest that in patients with RAS the frequency of silent CAD is high and cannot be predicted on clinical grounds alone, therefore coronary angiography should be routinely recommended in the same setting.

Long-term inhibition of the central alpha(2B)-adrenergic receptor gene via recombinant AAV-delivered antisense in hypertensive rats.

BACKGROUND: Salt-induced hypertension is mediated via the alpha(2B)-adrenergic receptor (AR) subtype. In alpha(2B)-AR gene knockout mice, blood pressure (BP) does not rise with salt loading, and in rats with salt-induced hypertension, BP decreases transiently with antisense (AS) treatment targeting the alpha(2B)-AR gene. The present experiments were designed to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA via adeno-associated virus (AAV) to prolong alpha(2B)-AR inhibition and hence reversal of salt-dependent hypertension. METHODS: A recombinant AAV (rAAV) vector preparation encoding the alpha(2B)-AS fragment (previously tested in vitro for inhibition of alpha(2B)-AR protein production in cells) and containing green fluorescence protein (GFP) for visualization was injected ICV into subtotally nephrectomized, salt-fed rats. Control rats received rAAV-GFP (n = 8 per group). RESULTS: We observed that BP rose from a baseline of 120 +/- 10 to 184 +/- 12 mm Hg. Injection of rAAV-alpha(2B)-AS produced a 35 +/- 12 mm Hg fall in BP, lasting without evidence of diminishing for at least 16 days, whereas rAAV-GFP-injected rats showed a continued rise in BP. Rats treated with rAAV-alpha(2B)-AS treated had a 45% to 65% decrease in alpha(2B)-AR protein levels in key regulatory regions of the brain. Neither group had signs of immunologic response to the virus injection. CONCLUSIONS: These results indicate that our construct, when given by ICV means, could reach multiple sites of the central nervous system relevant to BP regulation and could safely inhibit the central alpha(2B)-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension.

Central plasmid antisense administration reduces blood pressure inhibiting alpha2B adrenoceptor gene expression in spontaneously hypertensive rats in vivo.

INTRODUCTION: The involvement of central alpha2B adrenoceptors (AR) in the maintenance of hypertension has been proven by a series of previous experiments, at least in a particular model of nephrogenic salt-induced hypertension. The aim of the present study was to investigate further the role of central alpha2B AR in hypertension by applying antisense technology in another experimental model, the spontaneously hypertensive rat (SHR). METHODS: Plasmid antisense DNA against the alpha2B gene was given by intracerebroventricular injection to salt-fed SHRs, while a control group received plasmid alone. RESULTS: There was a significant fall in blood pressure, by an average of 31 +/- 12 mmHg, within the first twenty hours after injection in the antisense group. On the first post-injection day the blood pressure fell from 204 +/- 5.3 mmHg to 176.8 +/- 2.9 mmHg (p = 0.02). However, no significant changes in blood pressure were noticed in the plasmid group. Body-weight in both groups remained stable during the experiment. A study of frozen brain sections of SHRs after antisense DNA injection suggested that the nucleus tractus solitarii was one of the expression sites, while there was no histological evidence of tissue disruption. CONCLUSION: Central injection of antisense DNA targeting alpha2B mRNA in the genetic model of hypertension of the SHR seems to have a significant hypotensive effect, at least on the first day of injection. The nucleus tractus solitarii seems to be the primary area of action of central alpha2B AR in SHRs.

The A2B adenosine receptor protects against inflammation and excessive vascular adhesion.

Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.