Targeting insulin-like growth factor-1 receptor (IGF1R) for brain delivery of biologics.

The blood-brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non-invasive technologies targeting receptor-mediated pathways have been developed. In this study, three single-domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin-like growth factor-1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV-ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb-mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation. Compared to the brains perfused with the negative control A20.1-mFc, the brains perfused with anti-IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2-fold, p < .01) and the distribution volume (~4-fold, p < .01). The concentration curve for IGF1R4-mFc demonstrated a linear accumulation plateauing at approximately 400 µg (~1 µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post-ISBP confirmed the IGF1R4-mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1-mFc after a 5-min perfusion protocol. Systemic administration of IGF1R4-mFc fused with the non-BBB crossing analgesic peptide galanin (2 and 5 mg/kg) induced a dose-dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti-IGF1R sdAbs showed receptor-mediated brain uptake with pharmacologically effective parenchymal delivery of non-permeable neuroactive peptides.

Enhanced Delivery of Galanin Conjugates to the Brain through Bioengineering of the Anti-Transferrin Receptor Antibody OX26.

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having KDs of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood-brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB-crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high-affinity, selective antibody antagonist of the metabotropic glutamate receptor-1 (BBB-mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB-NiP). Both BBB-NiP and BBB-mGluR1 demonstrated a similar 20-fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo Comparative assessment of BBB-NiP and BBB-mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target-mediated removal of the mGluR1-engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose-dependent inhibition of mGluR-1-mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved in mGluR1-mediated pain processing. We demonstrate the feasibility of targeting central G-protein-coupled receptors using a BBB-crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.-Webster, C. I., Caram-Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

A Novel Antigen Design Strategy to Isolate Single-Domain Antibodies that Target Human Nav1.7 and Reduce Pain in Animal Models.

Genetic studies have identified the voltage-gated sodium channel 1.7 (Nav1.7) as pain target. Due to the ineffectiveness of small molecules and monoclonal antibodies as therapeutics for pain, single-domain antibodies (VHHs) are developed against the human Nav1.7 (hNav1.7) using a novel antigen presentation strategy. A 70 amino-acid peptide from the hNav1.7 protein is identified as a target antigen. A recombinant version of this peptide is grafted into the complementarity determining region 3 (CDR3) loop of an inert VHH in order to maintain the native 3D conformation of the peptide. This antigen is used to isolate one VHH able to i) bind hNav1.7, ii) slow the deactivation of hNav1.7, iii) reduce the ability of eliciting action potentials in nociceptors, and iv) reverse hyperalgesia in in vivo rat and mouse models. This VHH exhibits the potential to be developed as a therapeutic capable of suppressing pain. This novel antigen presentation strategy can be applied to develop biologics against other difficult targets such as ion channels, transporters and GPCRs.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: a role for angiotensin type 1 receptor.

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT1-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage.

Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor.

The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.

Transient receptor potential melastatin 7 (TRPM7) cation channels, magnesium and the vascular system in hypertension.

Decreased Mg(2+) concentration has been implicated in altered vascular reactivity, endothelial dysfunction and structural remodeling, processes important in vascular changes and target organ damage associated with hypertension. Unlike our knowledge of other major cations, mechanisms regulating cellular Mg(2+) handling are poorly understood. Until recently little was known about protein transporters controlling transmembrane Mg(2+) influx. However, new research has uncovered a number of genes and proteins identified as transmembrane Mg(2+) transporters, particularly transient receptor potential melastatin (TRPM) cation channels, TRPM6 and TRPM7. Whereas TRPM6 is found primarily in epithelial cells, TRPM7 is ubiquitously expressed. Vascular TRPM7 has been implicated as a signaling kinase involved in vascular smooth muscle cell growth, apoptosis, adhesion, contraction, cytoskeletal organization and migration, and is modulated by vasoactive agents, pressure, stretch and osmotic changes. Emerging evidence suggests that vascular TRPM7 function might be altered in hypertension. The present review discusses the importance of Mg(2+) in vascular biology in hypertension and focuses on transport systems, mainly TRPM7, that might play a role in the control of vascular Mg(2+) homeostasis. Elucidation of the relationship between the complex systems responsible for regulation of Mg(2+) homeostasis, the role of TRPM7 in vascular signaling, and the cardiovascular impact will be important for understanding the clinical implications of hypomagnesemia in cardiovascular disease.

Differentiation of Adipose-Derived Stem Cells into Vascular Smooth Muscle Cells for Tissue Engineering Applications.

Synthetic grafts have been developed for vascular bypass surgery, however, the risks of thrombosis and neointimal hyperplasia still limit their use. Tissue engineering with the use of adipose-derived stem cells (ASCs) has shown promise in addressing these limitations. Here we further characterized and optimized the ASC differentiation into smooth muscle cells (VSMCs) induced by TGF-ß and BMP-4. TGF-ß and BMP-4 induced a time-dependent expression of SMC markers in ASC. Shortening the differentiation period from 7 to 4 days did not impair the functional property of contraction in these cells. Stability of the process was demonstrated by switching cells to regular growth media for up to 14 days. The role of IGFBP7, a downstream effector of TGF-ß, was also examined. Finally, topographic and surface patterning of a substrate is recognized as a powerful tool for regulating cell differentiation. Here we provide evidence that a non-woven PET structure does not affect the differentiation of ASC. Taken together, our results indicate that VSMCs differentiated from ASCs are a suitable candidate to populate a PET-based vascular scaffolds. By employing an autologous source of cells we provide a novel alternative to address major issues that reduces long-term patency of currently vascular grafts.

Receptor and nonreceptor tyrosine kinases in vascular biology of hypertension.

PURPOSE OF REVIEW: Extensive evidence indicates that receptor tyrosine kinases and nonreceptor tyrosine kinases underlie vascular damage in hypertension. However, recent clinical studies using vascular endothelial growth factor (VEGF) receptor inhibitors (bevacizumab, axitinib) revealed the unexpected finding of increased blood pressure. Whether this is a generalized receptor tyrosine kinase phenomenon or a VEGF receptor-specific effect is unclear. The present review focuses on current findings regarding the role of tyrosine kinases and signaling in vascular pathobiology of hypertension. RECENT FINDINGS: Multiple complex and interacting signaling pathways activated by receptor and nonreceptor tyrosine kinases are upregulated and have been implicated in vascular alterations associated with high blood pressure. Experimental evidence suggests that receptor tyrosine kinase activation by direct ligand binding as well as by ligand-independent mechanisms through transactivation by G protein-coupled receptors plays a role in vascular signaling and cardiovascular diseases. SUMMARY: Cellular mechanisms and signaling pathways mediated by tyrosine kinases involved in hypertensive vascular damage are currently the subject of intensive investigation. The unexpected finding of hypertension as a side effect in patients treated with VEGF receptor inhibitors suggests that some tyrosine kinases negatively regulate vascular function. Further characterization of these processes will provide greater understanding of the role of tyrosine kinases in vascular pathobiology in hypertension and should provide new insights on novel therapeutic targets.

Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms.

The present study investigated the role of ROS (reactive oxygen species) and COX (cyclo-oxygenase) in ethanol-induced contraction and elevation of [Ca2+]i (intracellular [Ca2+]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1-800 mmol/l) induced contraction in endothelium-intact (EC50: 306+/-34 mmol/l) and endothelium -denuded (EC50: 180+/-40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [NG-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100 micromol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100 micromol/l], oxyhaemoglobin (NO scavenger, 10 micromol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1 micromol/l) increased ethanol-induced contraction. Tiron [O2- (superoxide anion) scavenger, 1 mmol/l] and catalase (H2O2 scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10 micromol/l), SC560 (selective COX-1 inhibitor, 1 micromol/l), AH6809 [PGF2alpha (prostaglandin F2alpha)] receptor antagonist, 10 micromol/l] or SQ29584 [PGH2(prostaglandin H2)/TXA2 (thromboxane A2) receptor antagonist, 3 micromol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O2- and H2O2. Ethanol induced a transient increase in [Ca2+]i, which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca2+ signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear.

Angiotensin(1-7) blunts hypertensive cardiac remodeling by a direct effect on the heart.

Angiotensin-converting enzyme 2 (ACE2) converts the vasopressor angiotensin II (Ang II) into angiotensin (1-7) [Ang(1-7)], a peptide reported to have vasodilatory and cardioprotective properties. Inactivation of the ACE2 gene in mice has been reported by one group to result in an accumulation of Ang II in the heart and an age-related defect in cardiac contractility. A second study confirmed the role of ACE2 as an Ang II clearance enzyme but failed to reproduce the contractility defects previously reported in ACE2-deficient mice. The reasons for these differences are unclear but could include differences in the accumulation of Ang II or the deficiencies in Ang(1-7) in the mouse models used. As a result, the roles of ACE2, Ang II, and Ang(1-7) in the heart remain controversial. Using a novel strategy, we targeted the chronic overproduction of either Ang II or Ang(1-7) in the heart of transgenic mice and tested their effect on age-related contractility and on cardiac remodeling in response to a hypertensive challenge. We demonstrate that a chronic accumulation of Ang II in the heart does not result in cardiac contractility defects, even in older (8-month-old) mice. Likewise, transgenic animals with an 8-fold increase in Ang(1-7) peptide in the heart exhibited no differences in resting blood pressure or cardiac contractility as compared to age-matched controls, but they had significantly less ventricular hypertrophy and fibrosis than their nontransgenic littermates in response to a hypertensive challenge. Analysis of downstream signaling cascades demonstrates that cardiac Ang(1-7) selectively modulates some of the downstream signaling effectors of cardiac remodeling. These results suggest that Ang(1-7) can reduce hypertension-induced cardiac remodeling through a direct effect on the heart and raise the possibility that pathologies associated with ACE2 inactivation are mediated in part by a decrease in production of Ang(1-7).

Regulation of the novel Mg2+ transporter transient receptor potential melastatin 7 (TRPM7) cation channel by bradykinin in vascular smooth muscle cells.

BACKGROUND: Transient receptor potential melastatin 7 (TRPM7) channels have been identified in the vasculature. However, their regulation and function remain unclear. METHODS: Here, we tested the hypothesis that bradykinin and its second messenger cAMP upregulate TRPM7, which stimulates activation of annexin-1 (TRPM7 substrate) and increases transmembrane Mg2+ transport and vascular smooth muscle cell (VSMC) migration. Human and rat VSMCs were studied. TRPM7 phosphorylation was assessed by immunoprecipitation:immunoblotting using antiphospho-serine/threonine and anti-TRPM7 antibodies. [Mg2+]i was measured by mag-fura-2. TRPM7 was downregulated by small interfering RNA and 2-aminoethoxydiphenyl borate. Annexin-1 activity was assessed by cytosol-to-membrane translocation. Cell migration and invasion, functional responses to bradykinin, were assessed in transwell chambers. RESULTS: Bradykinin increased expression of TRPM7 and annexin-1. TRPM7 was rapidly (5 min) phosphorylated on serine/threonine but not on tyrosine residues by bradykinin. [Mg2+]i was increased in bradykinin-stimulated cells (0.53 versus 0.68 mmol/l, basal versus bradykinin, P < 0.01). Annexin-1 activation was increased by bradykinin and inhibited by 2-aminoethoxydiphenyl borate. Although Hoe 140 (B2 receptor antagonist), U-73122 (phospholipase C inhibitor), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (c-Src inhibitor) and chelerythrine (protein kinase C inhibitor) blocked bradykinin actions, dibutyryl-c-AMP was without effect. In small interfering RNA-transfected and in 2-aminoethoxydiphenyl borate-treated cells, bradykinin-induced Mg2+ influx and VSMC migration were reduced. CONCLUSION: Our results demonstrate that bradykinin regulates TRPM7 and its downstream target annexin-1 through phospholipase C-dependent, protein kinase C-dependent and c-Src-dependent and cAMP-independent pathways; effects are mediated through bradykinin type 2 receptor; and bradykinin regulates VSMC [Mg2+]i and migration through TRPM7. These data identify TRPM7/annexin-1/Mg2+ as a novel pathway in bradykinin signaling.

Ethanol consumption increases blood pressure and alters the responsiveness of the mesenteric vasculature in rats.

Chronic ethanol consumption and hypertension are related. In the current study we investigated whether changes in reactivity of the mesenteric arterial bed could account for the increased blood pressure associated with chronic ethanol intake. Changes in reactivity to phenylephrine and acetylcholine were investigated in the perfused mesenteric bed from rats treated with ethanol for 2 or 6 weeks and their age-matched controls. Mild hypertension was observed in chronically ethanol-treated rats. Treatment of rats for 6 weeks induced an increase in the contractile response of endothelium-intact mesenteric bed to phenylephrine, but not denuded rat mesenteric bed. The phenylephrine-induced increase in perfusion pressure was not altered after 2 weeks' treatment with ethanol. Moreover, acetylcholine-induced endothelium-dependent relaxation was reduced by ethanol treatment for 6 weeks, but not 2 weeks. Pre-treatment with indometacin, a cyclooxygenase inhibitor, reduced the maximum effect induced by phenylephrine (Emax) in endothelium-intact mesenteric bed from both control and ethanol-treated rats. No differences in the Emax values for phenylephrine were observed between groups in the presence of indometacin. L-NNA, a nitric oxide (NO) synthase (NOS) inhibitor, increased the Emax for phenylephrine in endothelium-intact mesenteric bed from control rats but not from ethanol-treated rats. Levels of endothelial NOS (eNOS) mRNA were not altered by chronic ethanol consumption. However, chronic ethanol intake strongly reduced eNOS protein levels in the mesenteric bed. This study shows that chronic ethanol consumption increases blood pressure and alters the reactivity of the mesenteric bed. Moreover, the increased vascular response to phenylephrine observed in the mesenteric bed is maintained by two mechanisms: an increased release of endothelial-derived vasoconstrictor prostanoids and a reduced modulatory action of endothelial NO, which seems to be associated with reduced post-transcriptional expression of eNOS.

Effect of chronic ethanol consumption on endothelin-1 generation and conversion of exogenous big-endothelin-1 by the rat carotid artery.

The purpose of the present work was to investigate whether conversion of exogenous applied big-endothelin-1 (Big-ET-1) as well as the basal release and mRNA levels of endothelin-1 (ET-1) is altered by ethanol consumption in the rat carotid. The measurement of the contraction induced by Big-ET-1 served as an indicative of functional endothelin (ET)-converting enzyme (ECE) activity. Cumulative application of exogenous Big-ET-1 elicited a concentration-related contraction with the concentration-response curve shifted to the right when compared to ET-1. In endothelium-intact rings, phosphoramidon (1 mmol/l), a nonselective ECE/neutral endopeptidase (NEP) inhibitor, produced a rightward displacement of the concentration-response curves and reduced the maximal contractile response to Big-ET-1. However, in endothelium-denuded rings phosphoramidon reduced the maximum contraction for Big-ET-1 but did not alter the potency when compared to the curves obtained in the absence of the inhibitor. Ethanol consumption for 2, 6, or 10 weeks reduced the contractile effect elicited by Big-ET-1 in carotid rings with intact endothelium when compared to control or isocaloric rings. However, no differences on Big-ET-1-induced contraction were observed after endothelial denudation. On the other hand, ethanol consumption increased ET-1-induced contraction. Finally, chronic ethanol consumption did not alter either the mRNA levels for pre-pro-ET-1 nor the basal release of ET-1. The present findings show that chronic ethanol consumption does not alter the mRNA levels for ET-1 or its basal release in the rat carotid. Moreover, ethanol intake reduces the contraction induced by exogenously applied Big-ET-1 in carotid rings with intact endothelium, a fact that might be the result of a reduced conversion of this peptide by ECE on its mature active peptide ET-1.

Pharmacologic and molecular characterization of the vascular ETA receptor in the venomous snake Bothrops jararaca.

Endothelins (ETs) and sarafotoxins (SRTXs) are active isopeptides that have very similar structures and functions. All isoforms interact with two specific G-protein-coupled receptors, ET(A) and ET(B). To characterize functional vascular ET receptors in the poisonous snake, Bothrops jararaca, cumulative concentration-response curves to ETs and SRTXs were performed in isolated aortic rings, in the absence and presence of selective ET receptor antagonists. Vascular expression of ET receptor messenger RNA (mRNA) was evaluated by reverse transcriptase (RT) polymerase chain reaction (PCR) analysis, and a fragment of the ET(A) receptor was cloned and sequenced. In vivo, ET-1 induced a dose-dependent biphasic response on anesthetized B. jararaca snakes. In vitro, ET-1, SRTX-b, ET-3, SRTX-c, and IRL-1620 induced concentration-dependent vasoconstriction, with a potency order suggesting the presence of typical ET(A) receptors. BQ-123, a selective ET(A) antagonist, inhibited contractions induced by ET-1 and SRTX-b with expected negative log of the dissociation constant, K(B), (pK(B)) values for mixed ET(A)/ET(B) receptor populations. The nonselective ET(A)/ET(B) receptors antagonist, PD-142893, produced similar inhibition. The ET(B) antagonist, IRL-1038, potentiated contractile responses to SRTX-c. ET-1 and SRTX-c responses were also potentiated when aortic rings were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) plus indomethacin. Processing of the B. jararaca aortic first-strand complementary DNA, by RT-PCR with primers designed from the Gallus gallus ET(A) receptor sequence, enabled isolation, purification, cloning, and sequencing of a single band. The partial sequence of the B. jararaca ET(A) receptor showed a very high sequence similarity with ET(A) receptor sequences from chicken, rat, human, and Xenopus. In conclusion, vascular responses to SRTXs/ETs in the B. jararaca aorta are mediated predominantly, but not exclusively, by typical ET(A) receptors.

c-Src Inhibition Improves Cardiovascular Function but not Remodeling or Fibrosis in Angiotensin II-Induced Hypertension.

c-Src plays an important role in angiotensin II (Ang II) signaling. Whether this member of the Src family kinases is involved in the development of Ang II-induced hypertension and associated cardiovascular damage in vivo remains unknown. Here, we studied Ang II-infused (400 ng/kg/min) mice in which c-Src was partially deleted (c-Src+/-) and in wild-type (WT, c-Src+/+) mice treated with a c-Src inhibitor (CGP077675; 25 mg/kg/d). Ang II increased blood pressure and induced endothelial dysfunction in WT mice, responses that were ameliorated in c-Src+/- and CGP077675-treated mice. Vascular wall thickness and cross-sectional area were similarly increased by Ang II in WT and c-Src+/- mice. CGP077675 further increased cross-sectional area in hypertensive mice. Cardiac dysfunction (ejection fraction and fractional shortening) in Ang II-infused WT mice was normalized in c-Src+/- mice. Increased oxidative stress (plasma thiobarbituric acid-reactive substances, hydrogen peroxide, and vascular superoxide generation) in Ang II-infused WT mice was attenuated in c-Src-deficient and CGP077675-treated mice. Hyperactivation of vascular c-Src, ERK1/2 (extracellular signal-regulated kinase 1/2), and JNK (c-Jun N-terminal kinase) in hypertensive mice was normalized in CGP077675-treated and c-Src+/- mice. Vascular fibronectin was increased by Ang II in all groups and further augmented by CGP077675. Cardiac fibrosis and inflammation induced by Ang II were amplified in c-Src+/- and CGP-treated mice. Our data indicate that although c-Src downregulation attenuates development of hypertension, improves endothelial and cardiac function, reduces oxidative stress, and normalizes vascular signaling, it has little beneficial effect on fibrosis. These findings suggest a divergent role for c-Src in Ang II-dependent hypertension, where c-Src may be more important in regulating redox-sensitive cardiac and vascular function than fibrosis and remodeling.

Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage.

Functional characterization and expression of endothelin receptors in rat carotid artery: involvement of nitric oxide, a vasodilator prostanoid and the opening of K+ channels in ETB-induced relaxation.

We aimed to functionally characterize endothelin (ET) receptors in the rat carotid artery. mRNA and protein expressions of both ETA and ETB receptors, evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblotting, were detected in carotid segments. Immunohistochemical assays showed that ETB receptors are expressed in the endothelium and smooth muscle cells, while ETA receptors are expressed only in the smooth muscle cells. In endothelium-denuded vessels, levels of ETB receptor mRNA were reduced. Vascular reactivity experiments, using standard muscle bath procedures, showed that ET-1 induces contraction in endothelium-intact and -denuded carotid rings in a concentration-dependent manner. Endothelial removal enhanced ET-1-induced contraction. BQ123 and BQ788, selective antagonists for ETA and ETB receptors, respectively, produced concentration-dependent rightward displacements of the ET-1 concentration-response curves. IRL1620, a selective agonist for ETB receptors, induced a slight vasoconstriction that was abolished by BQ788, but not affected by BQ123. IRL1620-induced contraction was augmented after endothelium removal. ET-1 concentration dependently relaxed phenylephrine-precontracted rings with intact endothelium. The relaxation was augmented in the presence of BQ123, reduced in the presence of BQ788 and completely abolished after endothelium removal. IRL1620 induced vasorelaxation that was abolished by BQ788 and endothelium removal, but not affected by BQ123. Preincubation of intact rings with N(G)-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), indomethacin or tetraethylammonium (TEA) reduced IRL1620-induced relaxation. The combination of L-NAME, indomethacin and TEA completely abolished IRL1620-induced relaxation while sulfaphenazole did not affect this response. 4-aminopyridine (4-AP), but not apamin, glibenclamide or charybdotoxin, reduced IRL1620-induced relaxation. The major finding of this work is that it firstly demonstrated functionally the existence of both ETA and ETB vasoconstrictor receptors located on the smooth muscle of rat carotid arteries and endothelial ETB receptors that mediated vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and the activation of voltage-dependent K+ channels.

Contribution of the endothelin and renin-angiotensin systems to the vascular changes in rats chronically treated with ouabain.

Renin-angiotensin and endothelin systems are involved in the cardiovascular effects produced by treatment with ouabain. We recently demonstrated that the contractile response to phenylephrine is decreased in ouabain-treated rats. The present study investigated whether endothelin-1 (ET-1) and angiotensin II (Ang II) contributes to the vascular changes observed in rats chronically treated with ouabain. Wistar rats were treated with ouabain (8.0 microg day(-1), s.c. pellets for 5 weeks) alone or in combination with an endothelin type A receptor (ET(A)) antagonist, BMS182874 (40 mg kg(-1) day(-1), per gavage) or an angiotensin type 1 (AT(1)) receptor antagonist, losartan (15 mg kg(-1) day(-1), p.o.). Treatment with ouabain increased systolic blood pressure and treatment with either losartan or BMS182874 prevented the development of ouabain-induced hypertension. The sensitivity and maximal response for phenylephrine were reduced in aortic rings from ouabain-treated rats. Removal of the endothelium or in vitro exposure to an inhibitor of nitric oxide synthase (NOS), N-nitro-L-arginine methyl ester (L-NAME, 100 microM) increased the responses to phenylephrine, an effect that was more pronounced in aortas from ouabain-treated rats. Endothelial NOS protein (eNOS) expression was increased after ouabain treatment. Treatment with BMS182874, but not with losartan, prevented the effects of ouabain on the reactivity of phenylephrine and in eNOS protein expression. Gene expression of pre-pro-ET-1 and ET(A) receptors was increased in aortic rings from ouabain-treated rats. ET(B) receptor gene expression was not altered by ouabain treatment. In conclusion, our results suggest that endothelin and angiotensin systems play an important role in the development of ouabain-induced hypertension. However, ET-1, by activation of ET(A) receptors, but not Ang II, contributes to changes in vascular reactivity to phenylephrine induced by chronic treatment with ouabain.