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.

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.

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.

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.

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.

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.

Acute ethanol intake induces mitogen-activated protein kinase activation, platelet-derived growth factor receptor phosphorylation, and oxidative stress in resistance arteries.

In the present study, we investigated the role of angiotensin type I (AT1) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol.

Aldosterone signaling through transient receptor potential melastatin 7 cation channel (TRPM7) and its α-kinase domain.

We demonstrated a role for the Mg(2+) transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg(2+) and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg(2+)]i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg(2+) responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg(2+) influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg(2+)-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.

A non-damaging chemical amination protocol for poly(ethylene terephthalate) - application to the design of functionalized compliant vascular grafts.

Bioengineering approaches have been intensively applied to create small diameter vascular grafts using artificial materials. However, a fully successful, high performing and anti-thrombogenic structure has not been achieved yet. In this study, we present the first step of a process aiming at biofunctionalizing previously designed compliant polyethylene terephthalate (PET) scaffolds (Moreno et al., 2011). The main challenge of such a surface modification is to prevent the bulk polymer from any damage, so that it preserves the mechanical properties that the structures have been designed for. In that endeavor, an aminated long-chain polymer (polyvinylamine, PVAm) was used as an aminolysis reagent to get amine (-NH2) moieties only on the very surface of PET. Different reaction conditions were assayed, leading to a large range of amino group densities associated with slight variations of the planar tensile properties. These results were in stark contrast with those generated with a common small diamine substrate (ethylenediamine, EtDA), as the latter yielded a strong degradation of the mechanical properties for comparable amine densities. Tubular mechanical assays were then carried out on PVAm-functionalized PET scaffolds. The latter showed a compliance match with arteries under the chosen reaction conditions, as initially observed for pristine PET tubular scaffolds.

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.

Adipocytes produce aldosterone through calcineurin-dependent signaling pathways: implications in diabetes mellitus-associated obesity and vascular dysfunction.

We reported aldosterone as a novel adipocyte-derived factor that regulates vascular function. We aimed to investigate molecular mechanisms, signaling pathways, and functional significance of adipocyte-derived aldosterone and to test whether adipocyte-derived aldosterone is increased in diabetes mellitus-associated obesity, which contributes to vascular dysfunction. Studies were performed in the 3T3-L1 adipocyte cell line and mature adipocytes isolated from human and mouse (C57BL/6J) adipose tissue. Mesenteric arteries with and without perivascular fat and mature adipocytes were obtained from obese diabetic db/db and control db/+ mice. Aldosterone synthase (CYP11B2; mRNA and protein) was detected in 3T3-L1 and mature adipocytes, which secrete aldosterone basally and in response to angiotensin II (Ang II). In 3T3-L1 adipocytes, Ang II stimulation increased aldosterone secretion and CYP11B2 expression. Ang II effects were blunted by an Ang II type 1 receptor antagonist (candesartan) and inhibitors of calcineurin (cyclosporine A and FK506) and nuclear factor of activated T-cells (VIVIT). FAD286 (aldosterone synthase inhibitor) blunted adipocyte differentiation. In candesartan-treated db/db mice (1 mg/kg per day, 4 weeks) increased plasma aldosterone, CYP11B2 expression, and aldosterone secretion were reduced. Acetylcholine-induced relaxation in db/db mesenteric arteries containing perivascular fat was improved by eplerenone (mineralocorticoid receptor antagonist) without effect in db/+ mice. Adipocytes possess aldosterone synthase and produce aldosterone in an Ang II/Ang II type 1 receptor/calcineurin/nuclear factor of activated T-cells-dependent manner. Functionally adipocyte-derived aldosterone regulates adipocyte differentiation and vascular function in an autocrine and paracrine manner, respectively. These novel findings identify adipocytes as a putative link between aldosterone and vascular dysfunction in diabetes mellitus-associated obesity.

Ethanol induces vascular relaxation via redox-sensitive and nitric oxide-dependent pathways.

We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with l-NAME (non selective NOS inhibitor, 100 µmol/L), 7-nitroindazole (selective nNOS inhibitor, 100 µmol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, 1 µmol/L), glibenclamide (selective blocker of ATP-sensitive K(+) channels, 3 µmol/L) and 4-aminopyridine (selective blocker of voltage-dependent K(+) channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O(2)(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H(2)O(2)) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 µmol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 µmol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by l-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca(2+) concentration ([Ca(2+)]c), O(2)(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca(2+)]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity.

Adipocyte-derived factors regulate vascular smooth muscle cells through mineralocorticoid and glucocorticoid receptors.

Adipose tissue influences vascular function through adipocyte-derived factors, including components of the renin-angiotensin-aldosterone system. Molecular mechanisms underlying these phenomena are elusive. We investigated the role of adipocyte-derived factors on mitogen-activated protein kinases (MAPKs), proinflammatory status, apoptosis, and mitogenic signaling in vascular smooth muscle cells (VSMCs) and questioned whether these effects involve mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and angiotensin II type 1 receptor (AT(1)R). Cultured mouse VSMCs were exposed to adipocyte-conditioned medium (ACM) from differentiated 3T3-L1 adipocytes. ACM induced phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase, p38MAPK, and extracellular signal-regulated kinase 1/2 and increased expression of proinflammatory and proliferative markers in VSMCs. Eplerenone (MR antagonist), mifepristone (GR antagonist), and candesartan (AT(1)R antagonist) inhibited ACM-induced effects on extracellular signal-regulated kinase 1/2, p38MAPK, and proliferating cell nuclear antigen, without influencing apoptosis (Bax, Bcl, and caspase 3). Stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation was inhibited by mifepristone and candesartan but not by eplerenone. ACM-induced increase of fibronectin, vascular cell adhesion molecule 1, and plasminogen activator inhibitor 1 expression was blocked by MR and AT(1)R antagonism but not by GR inhibition. ACM has no effect on GR, MR, and AT(1)R expression. Our data show that adipocyte-derived factors influence MAPK signaling, leading to VSMC proinflammatory and profibrotic responses through distinct pathways. Although ACM stimulates p38MAPK and extracellular signal-regulated kinase 1/2 phosphorylation through MR, GR, and AT(1)R, activation of stress-activated protein kinase/c-Jun N-terminal kinase involves GR and AT(1)R. These findings suggest that adipocyte-derived factors regulate VSMC function through specific MAPKs linked to MR, GR, and AT(1)R, a posttranslational phenomenon, because ACM did not influence receptor expression. Such cross-talk between adipocytes and VSMCs may provide a potential molecular mechanism linking renin-angiotensin-aldosterone system, adipocytes, and vascular function.

Dysregulation of renal transient receptor potential melastatin 6/7 but not paracellin-1 in aldosterone-induced hypertension and kidney damage in a model of hereditary hypomagnesemia.

RATIONALE: Hyperaldosteronism, important in hypertension, is associated with electrolyte alterations, including hypomagnesemia, through unknown mechanisms. OBJECTIVE: To test whether aldosterone influences renal Mg(2+) transporters, (transient receptor potential melastatin (TRPM) 6, TRPM7, paracellin-1) leading to hypomagnesemia, hypertension and target organ damage and whether in a background of magnesium deficiency, this is exaggerated. METHODS AND RESULTS: Aldosterone effects in mice selectively bred for high-normal (MgH) or low (MgL) intracellular Mg(2+) were studied. Male MgH and MgL mice received aldosterone (350 µg/kg per day, 3 weeks). SBP was elevated in MgL. Aldosterone increased blood pressure and albuminuria and increased urinary Mg(2+) concentration in MgH and MgL, with greater effects in MgL. Activity of renal TRPM6 and TRPM7 was lower in vehicle-treated MgL than MgH. Aldosterone increased activity of TRPM6 in MgH and inhibited activity in MgL. TRPM7 and paracellin-1 were unaffected by aldosterone. Aldosterone-induced albuminuria in MgL was associated with increased renal fibrosis, increased oxidative stress, activation of mitogen-activated protein kinases and nuclear factor-NF-κB and podocyte injury. Mg(2+) supplementation (0.75% Mg(2+)) in aldosterone-treated MgL normalized plasma Mg(2+), increased TRPM6 activity and ameliorated hypertension and renal injury. Hence, in a model of inherited hypomagnesemia, TRPM6 and TRPM7, but not paracellin-1, are downregulated. Aldosterone further decreased TRPM6 activity in hypomagnesemic mice, a phenomenon associated with hypertension and kidney damage. Such effects were prevented by Mg(2+) supplementation. CONCLUSION: Amplified target organ damage in aldosterone-induced hypertension in hypomagnesemic conditions is associated with dysfunctional Mg(2+)-sensitive renal TRPM6 channels. Novel mechanisms for renal effects of aldosterone and insights into putative beneficial actions of Mg(2+), particularly in hyperaldosteronism, are identified.

Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR.

AIMS: We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells (VSMCs). Here, we investigated molecular mechanisms and biological responses of this phenomenon, focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor (PDGFR) in c-Src-regulated proinflammatory responses by aldosterone. METHODS AND RESULTS: Studies were performed in cultured VSMCs from Wistar-Kyoto (WKY) rats and caveolin-1 knockout (Cav 1(-/-)) and wild-type mice. Aldosterone stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae. Cholesterol depletion with methyl-ß-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target, Pyk2. Aldosterone effects were recovered by cholesterol reload. Aldosterone-induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs. PDGFR is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs. AG1296, a PDGFR inhibitor, prevented c-Src phosphorylation and translocation to cholesterol-rich fractions. Aldosterone induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs, responses that were inhibited an by cholesterol depletion, caveolin-1 deficiency, AG1296 and PP2, a c-Src inhibitor. Mineralocorticoid receptor (MR) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and PDGFR increased upon aldosterone stimulation, indicating MR-lipid raft/signalling association. CONCLUSION: We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via PDGFR.

Sphingosine-1-phosphate-induced inflammation involves receptor tyrosine kinase transactivation in vascular cells: upregulation in hypertension.

Sphingosine-1-phosphate (S1P), a multifunctional phospholipid, regulates vascular cell function. Whether S1P influences vascular inflammatory responses, particularly in hypertension, is unclear. We tested the hypothesis that S1P is a proinflammatory mediator signaling through receptor tyrosine kinase transactivation and that responses are amplified in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats (SHRSPs), a model in which we demonstrated Edg1 (S1P1 receptor) to be a candidate gene for salt-sensitive hypertension. Vascular smooth muscle cell from Wistar-Kyoto rats and SHRSPs were studied. S1P receptor subtypes, S1P1 and S1P2, were similarly expressed in Wistar-Kyoto rats and SHRSPs. S1P induced phosphorylation of epidermal growth factor receptor and platelet-derived growth factor and activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, with amplified effects in SHRSPs versus Wistar-Kyoto rats. Inhibition of epidermal growth factor receptor and platelet-derived growth factor (with AG1478 and AG1296, respectively) abolished S1P-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase in Wistar-Kyoto rats with variable effects in SHRSPs. Vascular smooth muscle cell inflammation was evaluated by expression of adhesion molecules and functional responses assessed by monocyte adhesion. S1P stimulated expression of intercellular adhesion molecule 1 and vascular cell adhesion protein 1 and promoted monocyte adhesion, particularly in SHRSP cells. S1P-mediated inflammation was blunted by AG1478 and AG1296 in SHRSP cells. VPC23019, a S1P1 receptor antagonist, inhibited S1P-induced mitogen-activated protein kinase phosphorylation, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression, and monocyte adhesion. Our data indicate that molecular processes underlying vascular inflammation and cell adhesion in SHRSPs involve S1P/S1P1 receptors and phosphorylation of receptor tyrosine kinases. We identify a novel pathway linking S1P/S1P1 receptors to specific proinflammatory signaling pathways through epidermal growth factor receptor and platelet-derived growth factor transactivation, a process that is upregulated in SHRSPs. Such molecular events may contribute to vascular inflammation in hypertension.

Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR.

The functional significance and regulation of NAD(P)H oxidase (Nox) isoforms by angiotensin II (Ang II) and endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs) from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was studied. Expression of Nox1, Nox2, and Nox4 (gene and protein) and NAD(P)H oxidase activity were increased in SHR. Basal NAD(P)H oxidase activity was blocked by GKT136901 (Nox1/4 inhibitor) and by Nox1 siRNA in WKY cells and by siNOX1 and siNOX2 in SHR. Whereas Ang II increased expression of all Noxes in WKY, only Nox1 was influenced in SHR. Ang II-induced NAD(P)H activity was inhibited by siNOX1 in WKY and by siNOX1 and siNOX2 in SHR. ET-1 upregulated Nox expression only in WKY and increased NAD(P)H oxidase activity, an effect inhibited by siNOX1 and siNOX2. Nox1 co-localized with Nox2 but not with Nox4, implicating association between Nox1 and Nox2 but not between Nox1 and Nox4. These data highlight the complexity of Nox biology in VSMCs, emphasising that more than one Nox member, alone or in association, may be involved in NAD(P)H oxidase-mediated •O(2)(-) production. Nox1 regulation by Ang II, but not by ET-1, may be important in •O(2)(-) formation in VSMCs from SHR.

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.