Joachim Hoyer

Expression and Function of Endothelial Ca2+-Activated K+ Channels in Human Mesenteric Artery A Single-Cell Reverse Transcriptase–Polymerase Chain Reaction and Electrophysiological Study In Situ

Ca2+-activated K+ (KCa) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase–polymerase chain reaction analysis in combination with the patch-clamp technique to characterize KCa channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial KCa channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non–cancer patients with inactive diverticulitis. Expression of the intermediate-conductance KCa channel (hIK1) was detected in non–cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding KCa currents, whereas respective KCa currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non–cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (P <0.05). However, KCa current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance KCa channel (hSlo) was detected in single ECs from CA patients. The increased KCa channel expression in CA patients resulted in a 2.7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (P <0.05). This increased expression and function of KCa channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.

Central arteriovenous anastomosis for the treatment of patients with uncontrolled hypertension (the ROX CONTROL HTN study): a randomised controlled trial

BACKGROUNDnHypertension contributes to cardiovascular morbidity and mortality. We assessed the safety and efficacy of a central iliac arteriovenous anastomosis to alter the mechanical arterial properties and reduce blood pressure in patients with uncontrolled hypertension.nnnMETHODSnWe enrolled patients in this open-label, multicentre, prospective, randomised, controlled trial between October, 2012, and April, 2014. Eligible patients had baseline office systolic blood pressure of 140 mm Hg or higher and average daytime ambulatory blood pressure of 135 mm Hg or higher systolic and 85 mm Hg or higher diastolic despite antihypertensive treatment. Patients were randomly allocated in a 1:1 ratio to undergo implantation of an arteriovenous coupler device plus current pharmaceutical treatment or to maintain current treatment alone (control). The primary endpoint was mean change from baseline in office and 24 h ambulatory systolic blood pressure at 6 months. Analysis was by modified intention to treat (all patients remaining in follow-up at 6 months). This trial is registered with ClinicalTrials.gov, number NCT01642498.nnnFINDINGSn83 (43%) of 195 patients screened were assigned arteriovenous coupler therapy (n=44) or normal care (n=39). Mean office systolic blood pressure reduced by 26·9 (SD 23·9) mm Hg in the arteriovenous coupler group (p<0·0001) and by 3·7 (21·2) mm Hg in the control group (p=0·31). Mean systolic 24 h ambulatory blood pressure reduced by 13·5 (18·8) mm Hg (p<0·0001) in arteriovenous coupler recipients and by 0·5 (15·8) mm Hg (p=0·86) in controls. Implantation of the arteriovenous coupler was associated with late ipsilateral venous stenosis in 12 (29%) of 42 patients and was treatable with venoplasty or stenting.nnnINTERPRETATIONnArteriovenous anastomosis was associated with significantly reduced blood pressure and hypertensive complications. This approach might be a useful adjunctive therapy for patients with uncontrolled hypertension.nnnFUNDINGnROX Medical.

Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells.

OBJECTIVEnCa(2+) mobilization plays an important role in endothelial function by stimulating Ca(2+)-dependent synthesis of vasodilating factors. In addition to inositol-1,4,5-trisphosphate (InsP(3)) mediated Ca(2+) mobilization, Ca(2+) release from ryanodine-sensitive pools and Ca(2+)-influx through TRP channels have been suggested to be important in endothelial Ca(2+)-signaling. However, the function and molecular identity of TRP channels and ryanodine receptors in human endothelium in situ are still elusive. We hypothesized that expression of ryanodine-receptors (RyR) and TRP channels differs between human endothelium in situ and in cultured cells.nnnMETHODSnBy combining single-cell RT-PCR and patch-clamp techniques, expression of RyR and TRP channels was determined in situ in endothelial cells of human mesenteric artery (HMAECs) obtained from patients undergoing bowel resection and in the endothelial cell line EA.hy926.nnnRESULTSnAt the single cell level, expression of RyR 3 was detected in 25 and 5% of HMAECs and EA.hy926 samples, respectively. Expression of the RyR 1 and 2 was not detected in either HMAECs or EA.hy926. In patch-clamp experiments in HMAECs, applications of caffeine (0.5 mM) induced sustained hyperpolarization mediated by activation of Ca(2+)-activated K channels. In EA.hy926, caffeine-induced hyperpolarization was not detected. Single HMAECs expressed the TRP genes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP gene in HMAECs in situ whereas TRP3 expression was rarely detected. EA.hy926 expressed only TRP1. In patch clamp experiments in HMAECs, Ca(2+)-store depletion activated non-selective cation currents leading to Ca(2+) entry.nnnCONCLUSIONSnOur findings suggest that, in addition to InsP(3) mediated Ca(2+) release, Ca(2+) release from ryanodine-sensitive stores mediated by RyR3 and Ca(2+) entry through TRP1 might represent important components of endothelial Ca(2+) signaling in situ and thereby of endothelial function in intact human blood vessels.

Mechanosensitive Cation Channels in Aortic Endothelium of Normotensive and Hypertensive Rats

In response to humoral and hemodynamic stimuli, vascular endothelium regulates vascular tone by releasing endothelium-derived vasoactive factors. Stretch-activated cation channels have been postulated to act as endothelial mechanosensors that respond to changes in hemodynamic forces. We report the presence of a nonselective (n=98) and K+-selective (n=53) stretch-activated channel in rat intact aortic endothelium and isolated aortic endothelial cells. The nonselective channel showed a permeability ratio for Na+, K+, and Ca2+ of 1:0.95:0.23 and was completely blocked by 50 micromol/L gadolinium, a blocker of stretch-activated channels. The K+-selective channel was selectively permeable for K+, with a K+-Na+ permeability ratio of 10.9:1. In whole-cell current recordings, hyposmotic cell swelling induced an increase in cell conductance. The swelling-induced current was completely blocked by 50 micromol/L gadolinium, showing that stretch-activated channels were activated by cell swelling and carry macroscopic cell currents. In a comparative study with normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), the K+-selective stretch-activated channel was observed in a 4.4-fold higher density in adult SHR compared with WKY. Also, in adult SHR, the stretch sensitivity of the nonselective channel was nearly twice as high as in WKY. In contrast, channel properties were unchanged in young SHR (5 to 6 weeks old) compared with age-matched WKY. These data suggest that stretch-activated channels are regulated in their sensitivity and density when subjected to increased hemodynamic forces such as in hypertension. Since the channels are capable of acting as endothelial mechanosensors, the altered channel properties might contribute to an altered mechanoreception in hypertension.

Pressure-activated cation channel in intact rat endocardial endothelium

OBJECTIVEnThe endocardial endothelium (EE) regulates myocardial performance in response to humoral and mechanical stimuli. In vascular endothelium mechanosensitive ion channels (MSC) act as mechanosensors for hemodynamic changes. In the present study we examined whether MSC are present in intact EE of rat papillary muscle segments and characteristics of MSC are altered in experimental hypertension.nnnMETHODSnMSC were investigated by the use of standard patch-clamp technique. For a comparative study, ion channel characteristics were determined in EE of two-kidney-one-clip rats and sham-operated controls.nnnRESULTSnWe identified a new class of MSC with a mean conductance of 21.8 +/- 4.4 (s.d.) pS for K+ and Na+ and of 4.1 +/- 1.5 pS for Ca2+. Channel activity was initiated by positive pipette pressure and blocked by negative pipette pressure. Channel open probability (Po) was characterized by its pressure sensitivity. Po increased from 0.06 at 10 mmHg to 0.37 and 0.55 at 20 mmHg and 30 mmHg, respectively. Gadolinium (20 microM), a blocker of MSC, completely inhibited channel activity. In some experiments activation of this pressure-activated channel (PAC) was followed by the opening of a Ca2(+)-dependent non-selective cation channel (NSC). This indicates that Ca2+ influx through PAC may be sufficient to increase intracellular Ca2+ concentration and thereby to activate neighboring NSC. In renovascular hypertension (2K1C), channel density of PAC was significantly increased compared to sham-operated controls. Channel density of NSC was not changed in 2K1C compared to sham-operated controls.nnnCONCLUSIONnA novel type of Ca2+ permeable MSC in intact EE of rat ventricular papillary muscle was identified, which is regulated by membrane pressure. PAC might be implicated in EE mechanotransduction by inducing an intracellular Ca2+ signal. Up-regulation of PAC density in EE from 2K1C might contribute to an altered mechanotransduction in hypertension.

Regulation of pressure-activated channel in intact vascular endothelium of stroke-prone spontaneously hypertensive rats.

The pressure-activated cation channel (PAC), a novel type of mechanosensitive channel, has been suggested to act as a mechanosensor in aortic endothelium. In experimental hypertension, PAC function was up-regulated in the established phase of high blood pressure. This association of altered PAC function and elevated arterial pressure suggests that PAC function is regulated by alterations in blood pressure. In the present study, we electrophysiologically investigated PAC function in intact endothelium of aorta (EA) and mesenteric artery (EMA) from stroke-prone spontaneously hypertensive rats (SHRSP), SHRSP after 4 weeks of treatment with quinaprilat (10 mg/kg/day), and normotensive Wistar-Kyoto (WKY) rats. In untreated SHRSP and WKY rats, systolic blood pressure (SBP) was 201+/-3 mm Hg and 142+/-3 mm Hg, respectively. In quinaprilat-treated SHRSP, SBP was lowered to 135+/-5 mm Hg. Apparent PAC density (percentage of patches with PAC activity) in EA of untreated SHRSP (63.7%+/-7.3%) was 2.4-fold higher than in WKY rats (26.0%+/-5.0%). In contrast, no significant PAC up-regulation was detected in EMA of SHRSP (15.7%+/-4.2%) compared with WKY rats (12.0%+/-3.9%). In EA of quinaprilat-treated normotensive SHRSP, PAC density (27.1%+/-5.2%) was lowered to levels found in normotensive WKY rats. Unitary conductance and pressure sensitivity of PAC were not altered in either hypertensive or normotensive rats. Taken together, hypertension-induced increases of endothelial PAC density can be completely reversed by antihypertensive therapy. The PAC up-regulation in EA was interpreted as a compensatory mechanism to enhance Ca2+-influx and subsequently the synthesis of vasodilatory factors. This mechanism is missing in EMA of SHRSP, which might contribute to high blood pressure in this rat model of severe genetic hypertension.

Central Iliac Arteriovenous Anastomosis for Uncontrolled Hypertension: One-Year Results From the ROX CONTROL HTN Trial

Creation of a central iliac arteriovenous anastomosis using a novel nitinol coupler device results in an immediate, significant reduction of blood pressure (BP). We present efficacy and safety findings at 12 months post-coupler insertion. This open-label, multicenter, prospective, randomized trial enrolled patients with a baseline office systolic BP ≥140 mmu2009Hg and average daytime ambulatory BP ≥135/85 mmu2009Hg. Subjects were randomly allocated to coupler implantation and continuing previous pharmacotherapy or to maintain previous treatment alone. At 12 months, 39 patients who had coupler therapy were included in the intention-to-treat analysis. Office-based systolic BP reduced by 25.1±23.3 mmu2009Hg (baseline, 174±18 mmu2009Hg; P<0.0001) post-coupler placement, and office diastolic BP reduced by 20.8±13.3 mmu2009Hg (baseline, 100±13 mmu2009Hg; P<0.0001). Mean 24-hour ambulatory BP reduced by 12.6±17.4/15.3±9.7 mmu2009Hg (P<0.0001 for both). In a prespecified subset of patients who failed to respond adequately to prior renal denervation, coupler therapy led to highly significant reduction in office systolic/diastolic BP (30.7/24.1 mmu2009Hg) and significant reduction in 24-hour ambulatory systolic/diastolic BP (12.4/14.4 mmu2009Hg) at 12 months (n=9). After coupler therapy, 14 patients (33%) developed ipsilateral venous stenosis; all were treated successfully with venous stenting. These findings confirm the importance of arterial mechanics in the pathophysiology of hypertension and support the clinical use of a central iliac arteriovenous anastomosis. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01642498.

[OP.LB.02.04] CENTRAL ILIAC ARTERIOVENOUS ANASTOMOSIS FOR UNCONTROLLED HYPERTENSION: 12 MONTH FOLLOW UP RESULTS OF THE ROX CONTROL HTN RANDOMISED CONTROLLED TRIAL.

Objective: Creation of a central iliac arteriovenous (AV) anastomosis using a novel nitinol coupler device results in an immediate, significant reduction of blood pressure (BP). We present efficacy and safety findings at 12 months post coupler insertion, extending the initial 6 month primary endpoint report (Lancet 2015). Design and method: This open-label, multicentre, prospective, randomised trial, enrolled patients with a baseline office systolic blood pressure (SBP) >140u200ammHg, average daytime ambulatory SBP > 135u200ammHg, and ambulatory diastolic blood pressure (DBP) > 85u200ammHg. Subjects were randomly allocated to AV coupler implantation and continuing previous pharmacotherapy, or to maintain previous treatment alone. Results: Eighty-three (43%) of 195 patients screened for eligibility were randomly allocated to AV coupler therapy (nu200a=u200a44) or control (nu200a=u200a39) groups. At 12 months, 39 patients who had AV coupler therapy were included in the intention to treat analysis. Office-based SBP reduced by 25.1u200a±u200a23.3u200ammHg (baseline 175u200a±u200a18u200ammHg, pu200a<u200a0·0001) post AV coupler placement, and office DBP reduced by 20.8u200a±u200a13.3u200ammHg (baseline 100u200a±u200a13u200ammHg, pu200a<u200a0·0001). Mean 24-hour ambulatory SBP reduced by 12.6u200a±u200a17.4u200ammHg (baseline 157u200a±u200a15u200ammHg, pu200a<u200a0·0001), post coupler treatment. Mean 24-hour ambulatory DBP reduced by 15.3u200a±u200a9.7u200ammHg (baseline 93u200a±u200a11u200ammHg, pu200a<u200a0·0001). (see Figure). Figure. No caption available. In a pre-specified subset of patients who had failed to respond adequately to prior renal denervation, AV coupler therapy lead to highly significant reduction in office SBP/DBP (30.7/24.1u200ammHg) and significant reduction in 24 hour ambulatory SBP/DBP (12.4/14.4u200ammHg) at 12 months follow up. In the 12 months following randomisation, the control population has experienced a single death, attributed to hypertensive heart failure, and 6 hospitalisations in 4 patients. Following AV coupler therapy, 14 patients (33%) developed ipsilateral venous stenosis; all were treated successfully with venous stenting. Conclusions: Office and ambulatory BP were substantially and durably reduced following coupler implantation. The control population experienced both hypertension-related morbidity and mortality, not observed in the treatment group. These findings confirm the importance of arterial mechanics in the pathophysiology of hypertension and support the clinical utility of a central iliac AV anastomosis.