Jorge Navarro-Dorado

Mechanisms involved in testosterone-induced vasodilatation in pig prostatic small arteries

AIMS Testosterone is beneficial to the cardiovascular system due to its direct coronary vasodilatory action and its circulatory deficiency is associated with coronary artery disease (CAD), which has been proposed as an extrinsic risk factor for benign prostatic hyperplasia (BPH). Therefore, the current study investigated the mechanisms involved in the testosterone-induced vasodilatation in pig prostatic small arteries. MAIN METHODS The testosterone vasoactive effects were assessed in small arterial rings mounted in microvascular myographs for isometric force recordings. KEY FINDINGS Testosterone and the non-aromatizable metabolite 4, 5alpha-dihydrotestosterone (DHT) evoked a similar concentration-dependent relaxation on noradrenaline (NA)-precontracted rings. Similar responses were obtained in preparations contracted with 60 mM K(+)-enriched physiological saline solution. Endothelium mechanical removal or pre-treatment with blockers of nitric oxide (NO) synthase, guanylate cyclase, aromatase activity, intracellular androgenic receptor (AR), 5alpha-reductase, prostanoid synthesis and K(+) channels, failed to modify the responses to testosterone. In Ca(2+)-free 124 mM KPSS, testosterone markedly inhibited in a concentration-dependent manner the contraction curve t degrees CaCl(2). In arteries pretreated with an L-type voltage-activated Ca(2+) channels (VOCCs) inhibitor, nifedipine, testosterone still relaxed noradrenaline-precontracted arteries. SIGNIFICANCE These data suggest that testosterone induces a direct vasodilatory action in pig prostatic small arteries independent of either endothelium, NO, prostanoids, aromatase or 5alpha-reductase activities, AR or K(+) channels. Such an effect is suggested to be produced via blockade of extracellular Ca(2+) entry through L-type VOCCs and non-L-type Ca(2+) channels. Testosterone-induced vasodilatation could be useful to prevent prostatic ischemia.

Role of neuronal voltage-gated K+ channels in the modulation of the nitrergic neurotransmission of the pig urinary bladder neck

As nitric oxide (NO) plays an essential role in the inhibitory neurotransmission of the bladder neck of several species, the current study investigates the mechanisms underlying the NO‐induced relaxations in the pig urinary bladder neck.

The complex regulation of TGF-β in cardiovascular disease

Transforming growth factor β (TGF-β1) is a pleiotropic cytokine with many and complex effects in cell and tissue physiology. This is made possible by a very complex and interwoven signaling system, whose regulation continues to be the focus of a growing line of research. This complex regulation translates to a key role in cardiovascular physiology, hemostasis, and the blood–vessel interface. In accordance with this, the TGF-β1 pathway appears to be deregulated in related disorders, such as atherosclerotic vascular disease and myeloproliferative syndromes. It is expected that the growing amount of experimental and clinical research will yield medical advances in the applications of knowledge of the TGF-β1 pathway to diagnosis and therapeutics.

Pharmacological basis and clinical evidence of dabigatran therapy

Dabigatran is an emerging oral anticoagulant which is a direct inhibitor of thrombin activity. It has been approved in the European Union and the United States of America for the prevention of thrombosis after major orthopedic surgery. It has also been approved by the American Food and Drug Administration and the European Medicines Agency for the prevention of stroke in chronic atrial fibrillation. Dabigatran provides a stable anticoagulation effect without any need to perform periodical laboratory controls. Of note, there is a growing amount of clinical evidence which shows its safety and efficacy. For these reasons, dabigatran may suppose a revolution in oral anticoagulation. However, two important limitations remain. First, it is contraindicated in patients with end-stage renal disease. Second, there is no evidence of the prevention of thrombosis in mechanical heart valves.

Overproduction of cyclo-oxygenase-2 (COX-2) is involved in the resistance to apoptosis in vascular smooth muscle cells from diabetic patients: a link between inflammation and apoptosis

Aims/hypothesisInflammation is a common feature in cardiovascular diseases, including diabetes mellitus. In addition to the well-known inflammatory role of cyclo-oxygenase-2 (COX-2), this protein has also been implicated in apoptosis resistance in tumour cells. Vascular smooth muscle cells (VSMC) from diabetic patients are also resistant to apoptosis because of an increased abundance of B cell lymphoma 2 protein (BCL2). In this work, we investigated whether overproduction of COX-2 was involved in the resistance to apoptosis in VSMC from diabetic patients.MethodsVSMC were obtained from internal mammary arteries from patients who had undergone coronary artery bypass graft surgery. Apoptosis was measured by DNA fragmentation, BCL2 degradation and cytochrome c release.ResultsApoptosis induced by C-reactive protein in cells from non-diabetic patients was mediated by COX-2. VSMC from diabetic patients showed higher basal levels of COX-2 compared with those from non-diabetic patients. Transfection of VSMC from non-diabetic patients with a plasmid containing COX-2 (also known as PTGS2) increased basal production of COX-2 and BCL2 and mimicked the resistance to apoptosis that occurs in diabetic patients. We also found a significant correlation (R = 0.846, p = 0.016) between COX-2 and BCL2 production in arterial rings from diabetic patients measured by confocal microscopy. However, inhibition of COX-2 production by small interfering RNA proved unable to reverse BCL2 production in diabetic VSMC.Conclusions/interpretationThese results suggest a link between inflammation (COX-2) and apoptosis resistance (BCL2) in the arteries of diabetic patients. This relationship is not causative and the common production of these two proteins may be co-regulated by shared regulatory elements in diabetes.

Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

Background We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-β1). Methodology/Principal Findings VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-β1 was able to reverse this effect. ASA (2 mM) induced TGF-β1 secretion; however it was unable to induce Smad activation. ASA increased p38MAPK phosphorylation in a TGF-β1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-β1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged. Conclusions/Significance In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-β1 and p38MAPK. Pre-surgical serum levels of TGF- β1 from bypass patients who took aspirin at antiplatelet doses did not change.

Mechanisms involved in the effects of endothelin-1 in pig prostatic small arteries

Since endothelin-1 (ET-1) is involved in prostatic disorders, the current study investigated the mechanisms underlying the ET-1-induced effects in pig prostatic small arteries. The experiments were performed in rings mounted in microvascular myographs containing physiological saline solution at 37oC for isometric force recordings. On basal tension, ET-1 (0.1-30 nM) evoked concentration-dependent contractions, which were enhanced by endothelium removal. ET-1 contractions were inhibited by blockade of endothelin ETA and ETB receptors, extracellular Ca2+ removal and blockade of voltage-dependent (L-type)- and non-voltage-dependent-Ca2+ channels. On endothelium intact rings precontracted with noradrenaline, the ETB endothelin receptor agonist BQ3020 promoted a concentration-dependent relaxation which was reduced by blockade of ETB receptors, nitric oxide synthase, guanylyl cyclase and prostanoids synthesis. Endothelium removal abolished its relaxant response and unmasked a BQ3020-induced contraction. Tetraethylammonium and 4-aminopyridine, blockers of non-selective K+ channels and voltage-dependent K+ (Kv) channels, respectively, inhibited the relaxations to BQ3020. Iberiotoxin, apamin and glibenclamide, blockers of large and small Ca2+-activated- and ATP-dependent- K+ channels, respectively, failed to modify these responses. These data suggest that ET-1 promotes contraction of pig prostatic small arteries by activating vascular smooth muscle contractile endothelin ETA and ETB receptors coupled to extracellular Ca2+ entry, via voltage-dependent (L-type)- and non-voltage-dependent Ca2+ channels, also being due to intracellular Ca2+ mobilization. In addition, a population of endothelial ETB receptors mediates vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and Kv channels.

A comparative study of α-adrenergic receptor mediated Ca2+ signals and contraction in intact human and mouse vascular smooth muscle

In many vascular smooth muscle cells, physiological and pharmacological agonists initiate oscillatory fluctuations in intracellular Ca(2+) to initiate and maintain vasoconstriction. These oscillations are supported by the underlying cellular ultrastructure, particularly the close apposition between the plasma membrane (PM) and superficial sarcoplasmic reticulum (SR), the so-called PM-SR junctions, which are important for SR Ca(2+) refilling. We hypothesize that the disappearance of PM-SR junctions during aging and/or disease is directly related to the disappearance of agonist-induced Ca(2+) oscillations. We compared phenylephrine-mediated Ca(2+) signals and contraction in human and murine smooth muscle cells in small mesenteric arteries and also employed electron microscopy to examine the cytoplasmic distribution of the SR. Phenylephrine elicited tonic contractions in both types of vessels, asynchronous Ca(2+) oscillations in the mouse mesenteric smooth muscle cells, but only single transient Ca(2+) signals in the human mesenteric smooth muscle cells. While nifedipine inhibited 90% of the phenylephrine-induced tonic contraction in mouse mesenteric arteries, it only slightly attenuated tonic contraction in human mesenteric arteries, although the nifedipine-resistant component was abolished by the Rho-kinase blocker 1-(5-Isoquinolinylsulfonyl)homopiperazine dihydrochloride (HA-1077). Furthermore, superficial SR was found to be abundant in the mouse vessels and many PM-SR junctions were observed, but the smooth muscle of human mesenteric arteries had far less peripheral SR and was almost devoid of PM-SR junctions. As PM-SR junctions are essential for the maintenance of Ca(2+) oscillations, the change in Ca(2+) signalling pattern in the relatively old human patients was due to impaired SR refilling.

Age-dependent defective TGF-beta1 signaling in patients undergoing coronary artery bypass grafting

BackgroundTransforming growth factor beta (TGF-β1) is a pleiotropic cytokine, which is deregulated in atherosclerosis; however the role of age in this process is unknown. We aimed to assess whether TGF-β1 signaling is affected by age.MethodsVascular smooth muscle cells (VSMC) were obtained from patients undergoing abdominal surgery. Levels of TGF-β1 were measured by ELISA in sera from 169 patients undergoing coronary artery bypass grafting (CABG). The p27 expression was determined by Western blot from internal mammary arteries (IMA) obtained from CABG patients (n = 13). In VSMC from these patients undergoing abdominal surgery, secretion of TGF-β1 was determined by ELISA of cell-conditioned media.ResultsIn VSMC from aged patients we observed a lower TGF-β1 secretion, measured as TGF-β1 concentration in cell conditioned medium (p < 0.001). This effect was correlated to an age-dependent decrease of p27 expression in IMA from aged CABG patients. In a similar manner, there was an age-dependent decrease of serum TGF-β1 levels in CABG patients (p = 0.0195).ConclusionsVSMC from aged patients showed a higher degree of cellular senescence and it was associated to a lower TGF-β1 secretion and signaling.

Calcium oscillations in human mesenteric vascular smooth muscle.

Phenylephrine (PE)-induced oscillatory fluctuations in intracellular Ca(2+) concentration ([Ca(2+)]i) of vascular smooth muscle have been observed in many blood vessels isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically-stimulated [Ca(2+)]i oscillations in human mesenteric artery smooth muscle. In arterial segments preloaded with Fluo-4 AM and mounted on a myograph on the stage of a confocal microscope, we observed PE-induced oscillations in [Ca(2+)]i, which initiated and maintained vasoconstriction. These oscillations present some variability, possibly due to compromised health of the tissue. This view is corroborated by our ultrastructural analysis of the cells, in which we found only (5 ± 2)% plasma membrane-sarcoplasmic reticulum apposition, markedly less than measured in healthy tissue from laboratory animals. We also partially characterized the oscillations by using the inhibitory drugs 2-aminoethoxydiphenyl borate (2-APB), cyclopiazonic acid (CPA) and nifedipine. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca(2+) and inositol 1,4,5-trisphosphate receptor (IP3R) in the maintenance of the Ca(2+) oscillations observed in human blood vessels.