Richard M. Cubbon

Piezo1 integration of vascular architecture with physiological force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

Orai1 and CRAC Channel Dependence of VEGF-Activated Ca2+ Entry and Endothelial Tube Formation

Rationale: Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown. Objective: The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation. Methods and Results: In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release–activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2′,5′-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry. Conclusions: The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.

Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance–induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance–mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance–related vascular disease.

Diabetes mellitus is associated with adverse prognosis in chronic heart failure of ischaemic and non-ischaemic aetiology

Background: It is unclear whether diabetes mellitus (DM) is an adverse prognostic factor in chronic heart failure (CHF) of ischaemic and non-ischaemic aetiology managed with contemporary evidence-based care. Methods: In total, 1091 outpatients with CHF with reduced ejection fraction were prospectively observed for a mean of 960 days. Total and cardiovascular mortality was quantified after accounting for potential confounders. Results: In total, 25.7% of patients had DM; this group was more likely to have CHF of ischaemic aetiology and was more symptomatic. Patients with DM received comparable medical- and device-based therapies, except for greater doses of loop diuretic. DM was associated with approximately doubled crude and adjusted risk of total and cardiovascular mortality. The association of diabetes with these outcomes in patients with ischaemic and non-ischaemic cardiomyopathies was of similar magnitude. Conclusions: In spite of advances in the management of CHF, DM remains a major adverse prognostic feature, irrespective of ischaemic/non-ischaemic aetiology.

Effects of insulin resistance on endothelial progenitor cells and vascular repair

Insulin resistance, a key feature of obesity, the metabolic syndrome and Type 2 diabetes mellitus, results in an array of metabolic and vascular phenomena which ultimately promote the development of atherosclerosis. Endothelial dysfunction is intricately related to insulin resistance through the parallel stimulatory effects of insulin on glucose disposal in metabolic tissues and NO production in the endothelium. Perturbations characteristic of insulin resistance, including dyslipidaemia, inflammation and oxidative stress, may jeopardize the structural or functional integrity of the endothelium. Recent evidence suggests that endothelial damage is mitigated by endogenous reparative processes which mediate endothelial regeneration. EPCs (endothelial progenitor cells) are circulating cells which have been identified as mediators of endothelial repair. Several of the abnormalities associated with insulin resistance, including reduced NO bioavailability, increased production of ROS (reactive oxygen species) and down-regulation of intracellular signalling pathways, have the potential to disrupt EPC function. Improvement in the number and function of EPCs may contribute to the protective actions of evidence-based therapies to reduce cardiometabolic risk. In the present article, we review the putative effects of insulin resistance on EPCs, discuss the underlying mechanisms and highlight potential therapeutic manoeuvres which could improve vascular repair in individuals with insulin resistance.

The Insulin-Like Growth Factor-1 Receptor Is a Negative Regulator of Nitric Oxide Bioavailability and Insulin Sensitivity in the Endothelium

OBJECTIVE In mice, haploinsufficiency of the IGF-1 receptor (IGF-1R+/−), at a whole-body level, increases resistance to inflammation and oxidative stress, but the underlying mechanisms are unclear. We hypothesized that by forming insulin-resistant heterodimers composed of one IGF-1Rαβ and one insulin receptor (IR), IRαβ complex in endothelial cells (ECs), IGF-1R reduces free IR, which reduces EC insulin sensitivity and generation of the antioxidant/anti-inflammatory signaling radical nitric oxide (NO). RESEARCH DESIGN AND METHODS Using a number of complementary gene-modified mice with reduced IGF-1R at a whole-body level and specifically in EC, and complementary studies in EC in vitro, we examined the effect of changing IGF-1R/IR stoichiometry on EC insulin sensitivity and NO bioavailability. RESULTS IGF-1R+/− mice had enhanced insulin-mediated glucose lowering. Aortas from these mice were hypocontractile to phenylephrine (PE) and had increased basal NO generation and augmented insulin-mediated NO release from EC. To dissect EC from whole-body effects we generated mice with EC-specific knockdown of IGF-1R. Aortas from these mice were also hypocontractile to PE and had increased basal NO generation. Whole-body and EC deletion of IGF-1R reduced hybrid receptor formation. By reducing IGF-1R in IR-haploinsufficient mice we reduced hybrid formation, restored insulin-mediated vasorelaxation in aorta, and insulin stimulated NO release in EC. Complementary studies in human umbilical vein EC in which IGF-1R was reduced using siRNA confirmed that reducing IGF-1R has favorable effects on NO bioavailability and EC insulin sensitivity. CONCLUSIONS These data demonstrate that IGF-1R is a critical negative regulator of insulin sensitivity and NO bioavailability in the endothelium.

Changing Characteristics and Mode of Death Associated With Chronic Heart Failure Caused by Left Ventricular Systolic Dysfunction A Study Across Therapeutic Eras

Background—Therapies for patients with chronic heart failure caused by left ventricular systolic dysfunction have advanced substantially over recent decades. The cumulative effect of these therapies on mortality, mode of death, symptoms, and clinical characteristics has yet to be defined. Methods and Results—This study was a comparison of 2 prospective cohort studies of outpatients with chronic heart failure caused by left ventricular systolic dysfunction performed between 1993 and 1995 (historic cohort: n=281) and 2006 and 2009 (contemporary cohort: n=357). In the historic cohort, 83% were prescribed angiotensin-converting enzyme inhibitors and 8.5% were prescribed &bgr;-adrenoceptor antagonists, compared with 89% and 80%, respectively, in the contemporary cohort. Mortality rates over the first year of follow-up declined from 12.5% to 7.8% between eras (P=0.04), and sudden death contributed less to contemporary mortality (33.6% versus 12.7%; P<0.001). New York Heart Association class declined between eras (P<0.001). QTc dispersion across the chest leads declined from 85 ms (SD, 2) to 34 ms (SD, 1) and left ventricular end-diastolic dimensions declined from 65 mm (SD, 0.6) to 59 mm (SD, 0.5) (both P<0.001). Conclusions—Survival has significantly improved in patients with chronic heart failure caused by left ventricular systolic dysfunction over the past 15 years; furthermore, sudden death makes a much smaller contribution to mortality, and noncardiac mortality is a correspondingly greater contribution. This has been accompanied by an improvement in symptoms and some markers of adverse electric and structural left ventricular remodeling.

Effects of Vitamin D on Cardiac Function in Patients With Chronic HF: The VINDICATE Study

Background Patients with chronic heart failure (HF) secondary to left ventricular systolic dysfunction (LVSD) are frequently deficient in vitamin D. Low vitamin D levels are associated with a worse prognosis. Objectives The VINDICATE (VitamIN D treatIng patients with Chronic heArT failurE) study was undertaken to establish safety and efficacy of high-dose 25 (OH) vitamin D3 (cholecalciferol) supplementation in patients with chronic HF due to LVSD. Methods We enrolled 229 patients (179 men) with chronic HF due to LVSD and vitamin D deficiency (cholecalciferol <50 nmol/l [<20 ng/ml]). Participants were allocated to 1 year of vitamin D3 supplementation (4,000 IU [100 μg] daily) or matching non−calcium-based placebo. The primary endpoint was change in 6-minute walk distance between baseline and 12 months. Secondary endpoints included change in LV ejection fraction at 1 year, and safety measures of renal function and serum calcium concentration assessed every 3 months. Results One year of high-dose vitamin D3 supplementation did not improve 6-min walk distance at 1 year, but was associated with a significant improvement in cardiac function (LV ejection fraction +6.07% [95% confidence interval (CI): 3.20 to 8.95; p < 0.0001]); and a reversal of LV remodeling (LV end diastolic diameter -2.49 mm [95% CI: -4.09 to -0.90; p = 0.002] and LV end systolic diameter -2.09 mm [95% CI: -4.11 to -0.06 p = 0.043]). Conclusions One year of 100 μg daily vitamin D3 supplementation does not improve 6-min walk distance but has beneficial effects on LV structure and function in patients on contemporary optimal medical therapy. Further studies are necessary to determine whether these translate to improvements in outcomes. (VitamIN D Treating patIents With Chronic heArT failurE [VINDICATE]; NCT01619891)

Insulin Resistance Impairs Circulating Angiogenic Progenitor Cell Function and Delays Endothelial Regeneration

OBJECTIVE Circulating angiogenic progenitor cells (APCs) participate in endothelial repair after arterial injury. Type 2 diabetes is associated with fewer circulating APCs, APC dysfunction, and impaired endothelial repair. We set out to determine whether insulin resistance adversely affects APCs and endothelial regeneration. RESEARCH DESIGN AND METHODS We quantified APCs and assessed APC mobilization and function in mice hemizygous for knockout of the insulin receptor (IRKO) and wild-type (WT) littermate controls. Endothelial regeneration after femoral artery wire injury was also quantified after APC transfusion. RESULTS IRKO mice, although glucose tolerant, had fewer circulating Sca-1+/Flk-1+ APCs than WT mice. Culture of mononuclear cells demonstrated that IRKO mice had fewer APCs in peripheral blood, but not in bone marrow or spleen, suggestive of a mobilization defect. Defective vascular endothelial growth factor–stimulated APC mobilization was confirmed in IRKO mice, consistent with reduced endothelial nitric oxide synthase (eNOS) expression in bone marrow and impaired vascular eNOS activity. Paracrine angiogenic activity of APCs from IRKO mice was impaired compared with those from WT animals. Endothelial regeneration of the femoral artery after denuding wire injury was delayed in IRKO mice compared with WT. Transfusion of mononuclear cells from WT mice normalized the impaired endothelial regeneration in IRKO mice. Transfusion of c-kit+ bone marrow cells from WT mice also restored endothelial regeneration in IRKO mice. However, transfusion of c-kit+ cells from IRKO mice was less effective at improving endothelial repair. CONCLUSIONS Insulin resistance impairs APC function and delays endothelial regeneration after arterial injury. These findings support the hypothesis that insulin resistance per se is sufficient to jeopardize endogenous vascular repair. Defective endothelial repair may be normalized by transfusion of APCs from insulin-sensitive animals but not from insulin-resistant animals.

Human Exercise-Induced Circulating Progenitor Cell Mobilization Is Nitric Oxide-Dependent and Is Blunted in South Asian Men

Objective—Circulating progenitor cells (CPC) have emerged as potential mediators of vascular repair. In experimental models, CPC mobilization is critically dependent on nitric oxide (NO). South Asian ethnicity is associated with reduced CPC. We assessed CPC mobilization in response to exercise in Asian men and examined the role of NO in CPC mobilization per se. Methods and Results—In 15 healthy, white European men and 15 matched South Asian men, CPC mobilization was assessed during moderate-intensity exercise. Brachial artery flow-mediated vasodilatation was used to assess NO bioavailability. To determine the role of NO in CPC mobilization, identical exercise studies were performed during intravenous separate infusions of saline, the NO synthase inhibitor l-NMMA, and norepinephrine. Flow-mediated vasodilatation (5.8%±0.4% vs 7.9%±0.5%; P=0.002) and CPC mobilization (CD34+/KDR+ 53.2% vs 85.4%; P=0.001; CD133+/CD34+/KDR+ 48.4% vs 73.9%; P=0.05; and CD34+/CD45− 49.3% vs 78.4; P=0.006) was blunted in the South Asian group. CPC mobilization correlated with flow-mediated vasodilatation and l-NMMA significantly reduced exercise-induced CPC mobilization (CD34+/KDR+ −3.3% vs 68.4%; CD133+/CD34+/KDR+ 0.7% vs 71.4%; and CD34+/CD45− −30.5% vs 77.8%; all P<0.001). Conclusion—In humans, NO is critical for CPC mobilization in response to exercise. Reduced NO bioavailability may contribute to imbalance between vascular damage and repair mechanisms in South Asian men.