Biomedical subjects

Martin Gosling

Publications and source records attributed to Martin Gosling.

12 recordsLinked to original sources

PubMed2006

The potassium channel opener levcromakalim causes expansive remodelling of experimental vein grafts.

BACKGROUND: Maintenance of luminal area is essential for the optimal performance of venous bypass grafts. However, injury and response to the arterial circulation evoke vascular remodelling that favors intimal hyperplasia, with luminal encroachment and inward remodelling. Potassium channel-opening drugs reduce tissue workload and peripheral vascular resistance and through these mechanisms could favor outward or expansive remodelling of vein grafts. We tested the hypothesis that levcromakalim, a potassium channel opener, would enhance expansive remodelling in vein grafts. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in 33 rats with vena cava-to-aorta bypass grafts. Drugs were administered via osmotic pump for 7 days after surgery. Half the cohort had bromodeoxyuridine (BrdU) infused at day 6. Morphometric analysis was conducted of pressure perfusion-fixed grafts harvested at 1 week and 4 weeks. RESULTS: At 1 week, lumen area was similar in both groups (1.82 +/- 0.39 mm(2) placebo vs 1.85 +/- 0.36 mm(2) levcromakalim), although medial cell density and BrdU staining were significantly increased in the placebo group. At 4 weeks, lumen area was unchanged in the placebo group (1.88 +/- 0.51 mm(2)) but had increased to 2.32 +/- 0.46 mm(2) in the levcromakalim group (P = .039 vs 1 week), with a very significant reduction in the intimal area (levcromakalim, 0.06 +/- 0.02 mm(2) vs placebo, 0.33 +/- 0.17 mm(2); P = .001). CONCLUSIONS: Early, short-term treatment with levcromakalim favors expansive remodelling of experimental vein grafts to mimic the effect of external stenting. This expansive remodelling was associated with a reduction in medial cell proliferation at 1 week. CLINICAL RELEVANCE: Critical limb ischemia can be treated by bypass surgery or angioplasty, but inward remodelling with restenosis is a common problem. There has been little previous experimental work to identify treatments associated with expansive remodelling, which would increase the chances of vessel patency. Here, in a randomized trial, we show that short-term treatment with a potassium channel opener (a class of drug that can be used to treat hypertension) results in strong, expansive remodelling, with increases the lumen area and graft size of experimental vein grafts by >25%.

Animals↗

PubMed2005

Determining the functional role of TRPC channels in primary cells.

Although the TRPC members of the mammalian transient receptor potential TRP cation channel family were the first to be described in 1995, the depth of knowledge of TRPC channels has fallen behind that of their counterparts in the TRPV and TRPM subfamilies in the intervening years. The complexities and controversies of TRPC channel composition and regulation have hindered their progress as therapeutic targets in the drug discovery environment to date, however embracing these challenges as opportunities may bring TRPC channels to the forefront of the discovery of novel therapies for many diseases. These challenges and opportunities of exploring TRPC channels as therapeutic targets are highlighted and discussed in this review with respect to respiratory diseases.

Alternative Splicing↗

PubMed2005

Therapeutic scope of modulation of non-voltage-gated cation channels.

Although widely regarded as attractive drug targets, less than a tenth of known ion channels are currently commercially exploited as therapeutic targets. Historically, drug discovery efforts on ion channel targets have been encumbered by a lack of molecular and structural information, sub-optimal screening technologies and a paucity of discriminating pharmacological tools. Although challenges remain, recent scientific and technological advances in the area of ion channel research and screening offer the exciting prospect of a new, more-predictive era of ion channel drug discovery. In this article, focusing primarily on non voltage gated cation channels, we describe the continuing evolution of approaches to ion channel drug discovery, highlight recent developments in the ion channel field and consider their potential impact on discovering and ascribing function to ion channel targets. We discuss the renaissance of known ion channel targets, such as nicotinic acetylcholine receptors and calcium-activated potassium channels, as well as the emergence of the transient receptor potential (TRP) channels as a gene family of cation channels with broad therapeutic potential.

Animals↗

PubMed2005

TRP channels in airway smooth muscle as therapeutic targets.

Cation channels are of fundamental importance in regulating the function of airway smooth cells especially bronchoconstriction in response to spasmogens, and are therefore key players in the pathogenesis of asthma. To date, the identity of these cation channels remains a mystery. However, the recently emerged transient receptor potential (TRP) cation channel family has provided several promising channel candidates. The identification of the key TRP channels involved in regulating airway smooth muscle contractility, and therefore airway tone, could provide new and exciting prospects for the development of novel therapies for the treatment of airway diseases such as asthma.

Animals↗

PubMed2004

Therapeutic scope of modulation of non-voltage-gated cation channels.

Although widely regarded as attractive drug targets, less than a tenth of known ion channels are currently commercially exploited as therapeutic targets. Historically, drug discovery efforts on ion channel targets have been encumbered by a lack of molecular and structural information, sub-optimal screening technologies and a paucity of discriminating pharmacological tools. Although challenges remain, recent scientific and technological advances in the area of ion channel research and screening offer the exciting prospect of a new, more-predictive era of ion channel drug discovery. In this article, focusing primarily on non-voltage-gated cation channels, we describe the continuing evolution of approaches to ion channel drug discovery, highlight recent developments in the ion channel field and consider their potential impact on discovering and ascribing function to ion channel targets. We discuss the renaissance of known ion channel targets, such as nicotinic acetylcholine receptors and calcium-activated potassium channels, as well as the emergence of the transient receptor potential (TRP) channels as a gene family of cation channels with broad therapeutic potential.

Animals↗

PubMed2004

Shear stress-induced shedding of soluble intercellular adhesion molecule-1 from saphenous vein endothelium.

Within 6 h, shear stress upregulated intercellular adhesion molecule-1 (ICAM-1) (two- to four-fold, P<0.001) and induced matrix metalloproteinase-2 (MMP-2) in cultured human saphenous vein endothelial cells. By 8 h endothelial ICAM-1 levels returned to baseline, with concomitant increase in soluble ICAM-1 (sICAM-1) (P<0.001) and MMP-9 had been induced. Inclusion of a hydroxamate metalloproteinase inhibitor partially reversed the effects on ICAM-1 and sICAM-1 at 8 h, whereas TIMP-1, -2 or -3 had no effect. MMP-9, but not MMP-2, co-immunoprecipitated with ICAM-1. sICAM-1 was processed distal to Arg441, indicating that MMP-9, docking to ICAM-1, contributes to sICAM-1 shedding and attenuation of the shear stress-induced upregulation of ICAM-1.

Endothelium, Vascular↗

PubMed2004

Activation of human TRPC6 channels by receptor stimulation.

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K(0.5) of approximately 10 microm and a Hill coefficient of 3-4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30-300 microm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 microm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.

Calcium Channels↗

PubMed2004

Flow-dependent increase of ICAM-1 on saphenous vein endothelium is sensitive to apamin.

The potassium channel blocker tetraethylammonium blocks the flow-induced increase in endothelial ICAM-1. We have investigated the subtype of potassium channel that modulates flow-induced increased expression of ICAM-1 on saphenous vein endothelium. Cultured human saphenous vein endothelial cells (HSVECs) or intact saphenous veins were perfused at fixed low and high flows in a laminar shear chamber or flow rig, respectively, in the presence or absence of potassium channel blockers. Expression of K(+) channels and endothelial ICAM-1 was measured by real-time polymerase chain reaction and/or immunoassays. In HSVECs, the application of 0.8 N/m(2) (8 dyn/cm(2)) shear stress resulted in a two- to fourfold increase in cellular ICAM-1 within 6 h (P < 0.001). In intact vein a similar shear stress, with pulsatile arterial pressure, resulted in a twofold increase in endothelial ICAM-1/CD31 staining area within 1.5 h (P < 0.001). Both increases in ICAM-1 were blocked by inclusion of 100 nM apamin in the vein perfusate, whereas other K(+) channel blockers were less effective. Two subtypes of small conductance Ca(2+)-activated K(+) channel (selectively blocked by apamin) were expressed in HSVECs and vein endothelium (SK3>SK2). Apamin blocked the upregulation of ICAM-1 on saphenous vein endothelium in response to increased flow to implicate small conductance Ca(2+)-activated K(+) channels in shear stress/flow-mediated signaling pathways.

Apamin↗

PubMed2003

F-actin capping (CapZ) and other contractile saphenous vein smooth muscle proteins are altered by hemodynamic stress: a proteonomic approach.

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.

Actin Cytoskeleton↗

PubMed2002

Maternal and postnatal vitamin D ingestion influences rat aortic structure, function and elastin content.

OBJECTIVES: Subtle impairment of fetal nutrition appears to predict hypertension and atherosclerosis in adults. It has been hypothesised that impaired aortic elastogenesis is the initiating step in adult hypertension and aortic aneurysms. Vitamin D has been shown to inhibit elastin synthesis by cultured smooth muscle cells. Here we have investigated, in rats, the hypothesis that increased exposure to vitamin D during gestation and in the postnatal period alters aortic elastin content and aortic function. METHODS: Nine breeding pairs of Sprague-Dawley rats were allocated to one of three diets containing 3000 (control group), 6000 (low dose) or 12,000 (high dose) IU/kg vitamin D during pregnancy and lactation. Male pups were continued on the same diet until 6 weeks of age. Aortic elastin content was assessed by measuring desmosine+isodesmosine content using capillary zone electrophoresis. Transverse aortic sections were used for quantification of elastic lamellae and morphometric analysis. The contractility of aortic rings was assessed in an organ bath preparation. RESULTS: The desmosine+isodesmosine content of the abdominal aorta of 6-week-old male pups, was 14.1, 10.0 and 10.1 nmol/mg dry weight in the control (n=20), low- (n=23) and high-dose (n=15) groups, respectively (P=0.007). The median number of elastic lamellae of the distal thoracic aorta was 8.25, 7.13 and 6.88 in the control, low-dose and high-dose groups, respectively (P<0.001). There were no significant differences in aortic cross-sectional areas or media:adventitia ratios. The mean peak tension of aortic rings, in response to phenylephrine, was 1.3 g, 1.12 g and 0.87 g in the control, low- and high-dose groups respectively (P=0.002). CONCLUSION: In rats, exposure to increased amounts of vitamin D during gestation and early life results in a reduction of aortic elastin content, number of elastic lamellae in the aorta and force generation in aortic rings.

Animal Nutritional Physiological Phenomena↗

PubMed2002

Circumferential stretching of saphenous vein smooth muscle enhances vasoconstrictor responses by Rho kinase-dependent pathways.

OBJECTIVE: Surgical preparation and/or pulsatile arterial perfusion of saphenous vein increases the sensitivity of vein rings to calcium mobilising agonists such as phenylephrine. We have investigated the mechanism(s) underlying this effect. METHODS: We have used an ex vivo flow circuit, with simulated arterial or venous flows (mean pressure 100 and 20 mmHg, respectively), to investigate the sensitivity of human saphenous vein to phenylephrine, 5-hydroxytryptamine (5-HT) and KCl, using organ chamber pharmacology. RESULTS: After 90 min of pulsatile arterial perfusion the mean maximum tension induced by KCl had increased from 4.7 to 11.1 g (n=5), by phenylephrine had increased from 4.4 to 10.2 g (n=8) and by 5-HT had increased from 4.4 to 6.7 g (n=10), all P<0.01. Phenylephrine did not augment the tension in vein rings maximally precontracted with KCl (n=4). The EC(50) for KCl was unchanged after pulsatile arterial perfusion (n=5), but for phenylephrine and 5-HT there were significant reductions from 14+/-5 to 2+/-1 microM (n=8) and from 1.0+/-0.4 to 0.20+/-0.06 microM (n=10), respectively. The rate of contraction (in response to 3 microM phenylephrine) increased from 0.11 g/min to 0.37 g/min, P<0.02, after arterial perfusion (n=4). These changes in contractile properties (to phenylephrine) were endothelium-independent, evident within 5 min of simulated arterial perfusion. The changes in contractile properties could be abrogated by external stenting of the vein (to attenuate circumferential deformation) or inclusion in the perfusate of a vasodilator, e.g., cromakalim (5 microM) or the selective Rho kinase inhibitor Y-27632 (20 microM). The heightened sensitivity and contractility to phenylephrine was maintained after inclusion of adenosine (100 microM), gadolinium (10 microM) or cycloheximide (10 microM) in the vein perfusate. CONCLUSIONS: The circumferential deformations imposed by simulated arterial perfusion alter the vasomotor responses of saphenous vein smooth muscle. These effects are independent of new protein synthesis or the activation of stretch activated cation channels. The Rho kinase pathway appears to mediate the signalling mechanisms leading to increased agonist-induced tension and the increased sensitivity to vasoconstrictors.

Adenosine↗

PubMed2002

Generation and characterisation of human saphenous vein endothelial cell lines.

Primary human endothelial cells have a finite life span in vitro. After 3-4 passages, they tend to de-differentiate and eventually reach senescence. This limits their use in studies of endothelial cell function. To overcome this, we have developed human saphenous vein endothelial cell lines (HSVEC lines). Two cell lines were produced by infection with pZipSVtsA58-U19 which encodes the simian virus 40 large T-antigen, and one cell line was obtained by transfection with pLXSN16E6E7, which encodes the human papillomavirus type 16 E6 and E7 genes. Two of the three HSVEC lines exhibited an extended life span in vitro and retained characteristic endothelial "cobblestone" morphology. These cell lines expressed the known endothelial markers CD31 and vascular endothelial cadherin, and were able to bind Ulex europaeus lectin I, but they did not retain the expression of von Willebrand factor. Furthermore, one cell line was able to functionally up-regulate the expression of intercellular adhesion molecule-1 in response to stimulation with tumor necrosis factor alpha and was also able to incorporate acetylated low-density lipoprotein. Our results suggest that this latter HSVEC line will provide a useful resource to investigate selected responses of the vascular endothelium to physiological and pathological situations.

Antibodies↗