This study tests the hypothesis that RMJH-111b, a novel drug candidate, can improve blood pressure (BP) in hypertensive adults without the gastrointestinal distress that is often experienced with oral magnesium (Mg) treatment. A Phase 1 / 2 study tested the clinical tolerability, safety and efficacy as well as pharmacokinetics of RMJH-111b softgels. Each softgel contains 110 mg elemental Mg as inverted micellar nanodroplets designed to enhance intestinal uptake and cell delivery of active constituents. Forty-one hypertensive people were screened, twenty-two qualified to be enrolled and twenty-one completed the 10 day inpatient trial. In the study, after a 7 day washout period, participants were admitted and had a 3 day run in on only placebo followed by a 7 day double blind treatment period. Fifteen people received active treatment with 440 mg of elemental Mg BID (a total of 880 mg daily) and six received placebo. Data collected include daily seated blood pressure as well as 24h ambulatory blood pressure (ABPM) measurements. In addition, ECG, routine laboratory tests including serum Mg levels and 24h urinary Mg excretion were performed on days 3 and 10.ABPM 24h mean blood pressure changes day, night and 24° are as follows: SBP day :-6.2, DBP day :-2.3, SBP night :-4.9 , DBP night :-1.3 , SBP 24° :-6.6 , DBP 24° :-2.8 , SBP+DBP 24° :-9.4. One individual was excluded because her SBP decreased from 155 to 108 mm Hg after five days on protocol, below the 110 mm SBP protocol exclusion criteria. No symptoms of hypotension were observed. A correlation was noted between 24h levels of urinary Mg excretion and the 24h ABPM BP reduction. There were no adverse effects in the study group. A trend correlating serum Mg levels increase and BP decrease across the study population was noted. Based on retroactive statistical analysis of data presented, if 100 people had been studied using the same protocol, a statistical significance with a p value.

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.

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.

Bradykinin participates in various hypertensive processes, exerted via its type 1 and type 2 receptors (BKR1 and BKR2). The aim of the study was to investigate BKR1 and BK2R gene expression in peripheral monocytes in patients with essential hypertension compared with healthy individuals. Seventeen hypertensive patients (9 males, age 56 ± 7 years) and 12 healthy individuals (7 males, age 55 ± 6) participated. Mononuclear cells isolated using anti-CD14+ antibodies and mRNAs of BKR1 and BKR2 were estimated by real-time quantitative reverse transcription-PCR. Both BKR1 and BKR2 showed significantly upregulated gene expression in the group of hypertensive patients. Specifically, BKR1 gene expression was 142.1 ± 42.2 in hypertensives versus 20.2 ± 8 in controls (P = 0.024) and BKR2 was 1222.2 ± 361.6 in hypertensives versus 259.5 ± 99.1 in controls (P = 0.038). Antihypertensive treatment resulted in a decrease in BKR1 (from 142.1 ± 42.2 to 55.2 ± 17.1, P = 0.065) and in BKR2 (from 1222.2 ± 361.6 to 256.8 ± 81.8, P = 0.014) gene expression. BKR1 and BKR2 gene expression on peripheral monocytes is upregulated in essential hypertension. This may lead to functional changes in monocytes and contribute to the development of target organ damage in hypertensive patients.

From the first description of its anatomy by T. Willis to the novel therapeutic manipulations, it is unanimously recognized that the sympathetic nervous system (SNS) holds a crucial role in cardiovascular homeostasis. The introduction of sophisticated techniques, as microneurography and regional norepinephrine spillover provided the evidence for the role of sympathetic overactivity in various cardiovascular disease entities. Sympathetic activation is common in patients with essential hypertension and contributes to initiation, maintenance and progression of the disease and it contributes to the manifestation of its major complications. A considerable body of evidence relates SNS overactivity with high sodium intake in experimental animals and humans and the underlying mechanisms have nowadays been elucidated. SNS activity is more pronounced in patients with resistant hypertension and there are several conditions that lead to this phenomenon, as older age, kidney disease, obesity and metabolic syndrome, mental stress and sleep apnea. SNS overactivity holds also a key physiopathological role in heart failure, acute coronary syndromes and arrhythmias. Moreover, inhibition of sympathetic overactivity by various means, including central SNS suppressing drugs, peripheral alpha- and beta- adrenergic receptor blockers, or novel approaches as renal sympathetic denervation have been used successfully in the treatment of all these disorders.

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 (alpha 2B-ARs) in normal human platelets.Methods: Sixteen healthy individuals were recruited as donors of normal blood from which platelets were isolated. The presence of alpha 2B-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 alpha 2B-AR antibodies and of the selective alpha 2B-AR antagonist ARC 239.Results: Pretreatment of human platelets with agents that selectively block alpha 2B-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 alpha 2B-Abs and ARC 239 respectively, from 72.2 +/- 1.9% to 25.5 +/- 4.3% and to 8.8 +/- 1.7% for epinephrine with alpha 2B-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 alpha 2B-Abs and ARC 239 respectively, p < 0.05 for all. Additionally, collagen/epinephrine closure time increased from 120.8 +/- 6.1 s to 189.5 +/- 39.5 s (p = 0.001).Conclusions: Our results reveal that contrary to previous knowledge, the alpha 2B-AR subtype does exist in platelets and is an important regulator of aggregation. Inhibition of alpha 2B-ARs in plateletsmay offer a novel therapeutic opportunity in the prevention of atherothrombotic events. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Purpose: 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 platelets from patients with Coronary Artery Disease (CAD) compared to normal individuals. Methods: Sixteen healthy individuals and 7 patients with CAD who were under dual antiplatelet therapy with clopidogrel 75mg and acetylsalicylic acid 100mg were included. Blood samples were obtained from which platelets were isolated. The presence of α2B-ARs in platelets was proven by Western blot analysis and real time PCR. In order to investigate their function, we performed light transmittance aggregometry by examining the inhibitory effects of specific α2B-AR antibodies. 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% for ADP with α2B-Abs, from 72.2±1.9% to 25.5±4.3% for epinephrine with α2B-Abs, and from 87±2.1% to 47.9±6.2% for arachidonic acid with α2B-Abs, p<0.05 for all. Patients with CAD showed a significantly reduced expression of the receptor in the platelets (149.6±100.8 in normals versus 32.1±17.7, p = 0.015). Notably, pretreatment with α2B –Abs resulted in a significant further reduction of platelet aggregation. The ADP-treated plasma showed a significant reduction in platelet aggregation after pre-treatment with α2B-Abs (42±5.8% for the control group versus 31.7±2.2% for the group with α2B-Abs pretreatment, p< 0.001). When arachidonic acid was used to induce aggregation, pre-treatment with α2B -Abs reduced platelet aggregation from 41.2±7.3% to 28.2±7.5%, p= 0.006). Similarly, the presence of α2B-Abs decreased platelet aggregation induced by epinephrine from 44.4±22.1% to 19.1±7.2% (p = 0.01). 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. Interestingly, even in patients with coronary artery disease who were receiving dual antiplatelet therapy with aspirin and clopidogrel, the inhibition of α2B-ARs had an additional antiaggregant effect.

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.

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 alpha(2)-adrenergic receptors located mostly in the brainstem. In recent years, the cloning and characterization of alpha(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 alpha(2A)-adrenergic receptors and the sympathoexcitatory alpha(2B)-adrenergic receptors, leading to a hyperadrenergic hypertensive state unrelated to volume overload.

The renin-angiotensin system (RAS) was extensively investigated and characterized throughout the first half of the 20th century. However, its contribution to the maintenance of high blood (BP) in essential hypertension and to the development of hypertensive cardiac complications remained under debate until the advent of the first pharmacologic probes capable of blocking its actions, namely the angiotensin receptor blocker (ARB) saralasin and the angiotensin-converting enzyme inhibitor (ACEI) teprotide in the early 1970’s. Using these probes, we could demonstrate that even in normal-renin and low-renin hypertension, blockade of the RAS produced a fall in BP. And this fall was maximized if the patient had been previously submitted to sodium depletion by diuretics or low salt diet, which might produce only a small and possibly insignificant BP fall by itself, but rendered the hypertension RAS-dependent and far more responsive to RAS blockade. In parallel, we had found that excess angiotensin II, either exogenous (in experimental animals) or endogenous, in various clinical settings, could produce significant cardiac and renal tissue damage, because the vasculature of these organs is particularly sensitive to the constricting effects of angiotensin. In particularly, angiotensin excess was shown to produce widespread foci of necrosis and scarring of the myocardium, leading to replacement of myocardial tissue by fibrotic tissue, eventually progressing to ischemic cardiomyopathy and heart failure. Early clinical trials found that treatment of heart failure with ACEIs or ARBs leads to significant hemodynamic improvement in terms of increased cardiac output, decreased peripheral arterial resistance, decreased heart rate, increased coronary blood flow and diminished myocardial oxygen consumption. Subsequent large randomized long-term outcome trials with ACEIs or ARBs, such as the HOPE, EUROPA, LIFE, CHARM, etc., confirmed the functional and structural benefits of these therapeutic approaches and have now established treatment with an ACEI or ARB as mandatory therapy for patients with ischemic heart disease, as well as congestive or chronic heart failure. It is also now universally accepted that initiation of RAS-suppressing therapy with an ACEI or ARB in patients with multiple cardiovascular risk factors, even not necessary including hypertension, offers long-term protection from ischemic heart disease, diastolic and systolic cardiac dysfunction, arrhythmias, as well as protection from renal insufficiency, cerebrovascular accidents and new onset type 2 diabetes mellitus. Drugs that inhibit the RAS now include, in addition to the ACEIs and ARBs, a new class, the direct renin inhibitors (DRIs), of which only aliskiren is commercially available to date. Several clinical trials have shown aliskiren to be equally effective to ACEIs and ARBs in terms of BP lowering, cardioprotection and nephroprotection. A large body of literature has now reported additional benefits of ACEI+ARB combination in selected patients, i.e., those with resistant hypertension on multidrug ATHENS CARDIOLOGY UPDATE 2010

The incidence of hypertension and attendant biological disorders (loss of arterial wall elasticity, endothelial dysfunction, insulin resistance) increases with age and so does the frequency of cardiovascular complications. Women have a lower incidence of hypertension and a later—by an average of 10 years—onset of cardiovascular complications. During the premenopausal years, women seem to be relatively protected from cardiovascular events, in part through the effects of estrogen on endothelial function and lipid profile. After menopause however, the incidence of cardiovascular events tends to become similar in both genders, and the severity of such events, in terms of morbidity and mortality, is actually higher in women. The role of hormone replacement for cardiovascular protection has been shown to offer no long-term benefits: indeed, despite improvement in surrogate endpoints (endothelial function, lipid profile), in long-term prospective randomized trials there was no advantage in outcomes, possibly because benefits are offset by the thrombogenic and carcinogenic properties of estrogen. Over age 65 the prevalence of hypertension can reach more than 50%. Systolic hypertension predominates, but is itself an important risk factor and marker of cardiovascular complications. Special considerations affecting choice of antihypertensive agents for elderly patients include knowledge of the normal pathophysiologic alterations of aging, assessment of coexisting diseases or target organ complications, consideration of the pharmacodynamics and kinetics of each class of antihypertensive agents and potential drug interactions when treating the frequently coexisting diseases, such as diabetes mellitus, chronic obstructive pulmonary disease, arthritis, etc. Contrary to previous assumptions, elderly hypertensives are not resistant to newer antihypertensive agents such as angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II type 1 receptor blockers (ARBs), and in fact appear to respond to lower doses with incidence of side-effects generally similar to that of younger individuals. ACEIs, ARBs and calcium channel blockers seem to have several advantages in comparison to older antihypertensive agents in this population, mostly because of lack of metabolic disturbances and of central nervous system-mediated side-effects (drowsiness, forgetfulness, fatigue etc) that adversely affect the quality of life of these patients. The newest antihypertensive drug class, direct renin inhibitors (DRI), whose first member, aliskiren, has recently become available, has not yet been studied specifically in elderly hypertensives. However, in view of its mechanism of action, it would be expected to have a pharmacologic profile essentially similar to that of ACEIs and ARBs. Evidence of the value of pharmacologic therapy in elderly patients with isolated systolic hypertension (ISH) was first provided in 1992 by the Systolic Hypertension in the Elderly Program (SHEP). However, as the BP treatment in those years was based mostly on thiazide diuretics, the benefits of BP lowering came at the price of significant adverse metabolic effects, including hypokalemic, hyperuricemia and increased rate of ATHENS CARDIOLOGY UPDATE 2010

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.

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 geneXirp2in Ang II–mediated cardiac remodeling was investigated.Methods and Results:Here we demonstrate that the MEF2A target geneXirp2(also known as cardiomyopathy associated gene 3 [CMYA3]) is an important effector of the Ang II signaling pathway in the heart.Xirp2belongs to the evolutionarily conserved, muscle-specific, actin-bindingXingene family and is significantly induced in the heart in response to systemic administration of Ang II. Initially, we characterized theXirp2promoter and demonstrate that Ang II activatesXirp2expression 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 theXirp2gene that resulted in a marked reduction in its expression in the heart. In the absence of Ang II, adultXirp2hypomorphic mice displayed cardiac hypertrophy and increased β myosin heavy chain expression. Strikingly,Xirp2hypomorphic 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.

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.