Endothelium-dependent Contracting Factor Research Articles

Endothelial Dysfunction and Enhanced Contractility in Microvessels From Ovariectomized Rats

Ovarian hormone loss increases reactive oxidative species, endothelial dysfunction, and cardiovascular disease. Because perivascular adipose tissue (PVAT) regulates endothelial function, we hypothesized that reactive oxidative species in PVAT mediate adverse microvascular effects of ovarian hormone deficiency. Rats were ovariectomized or sham operated and given vehicle or tempol for 6 weeks. Mesenteric resistance arterioles from ovariectomized compared with sham-operated rats had dysfunctional responses to acetylcholine (ACh) including decreased ACh-induced endothelium-dependent relaxation (50±6% versus 72±2%) and endothelium-dependent relaxation factor (17±4% versus 37±2%) and increased endothelium-dependent contracting factor (27±5% versus 9±3%). OVX rat mesenteric arterioles had increased contractions to the thromboxane/prostanoid receptor agonist U-46 619 (58±3% versus 40±5%) and increased reactive oxidative species (tempo-9-AC fluorescence) with U-46 619 (0.65±0.17 versus 0.14±0.06 Δ unit) or ACh (0.49±0.09 versus 0.09±0.05 Δ unit) and increased p22 phox protein expression (0.89±0.05 versus 0.18±0.04 Δ unit), whereas nitric oxide activity (DAF-FM [4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate] fluorescence) with ACh was reduced (0.39±0.1 versus 0.70±0.10 Δ unit). No differences were found in endothelium-dependent hyperpolarizing factor or contractile responses to phenylephrine. PVAT enhanced ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide only in sham-operated rats. Tempol prevented ovariectomy-induced endothelial dysfunction and restored the enhancing effects of PVAT on ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide in ovariectomized rat vessels, but both tempol and PVAT were required to normalize the enhanced U-46 619 contractions after ovariectomy. In conclusion, ovariectomy redirects endothelial responses from relaxation to contraction by reducing vascular nitric oxide, augmenting thromboxane/prostanoid receptor signaling, and attenuating the vasodilatory effects of PVAT, all of which were dependent on reactive oxidative species.

Abstract 500: Reactive Oxygen Species and Insufficiency of Nitric Oxide Mediate Enhanced Microvascular Contractility and Impaired Endothelium Dependent Relaxation in Mice With Renal Failure

Background: Cardiovascular disease (CVD) is common and major cause of death and disability in chronic kidney disease (CKD). Since CVD starts with endothelial dysfunction, we tested the hypothesis that reactive oxygen species (ROS) and insufficient nitric oxide (NO) contributed to microvascular contractility and endothelial dysfunction in C57BL/6 mice with normotensive reduced renal mass (RRM) 5/6 nephrectomy, a model of progressive CKD. Methods and Results: Mesenteric arterioles (MAs) were isolated from mice 3 months after sham-operation (Sham) or RRM (n=6/group) and were mounted on Mulvany-Halperin wire myograph, preconstricted with norepinephrine and relaxed with acetylcholine (ACh) for: endothelium-dependent relaxation (EDR); endothelium-dependent relaxation factor (EDRF; NOS-dependent relaxation); endothelium-dependent hyperpolarizing factor (EDHF; K + -channel dependent relaxation) and endothelium-independent relaxation (EIR; sodium nitroprusside). Contractions were tested to endothelium-dependent contracting factor (EDCF; ACh-induced contraction with blocked relaxation pathways); phenylephrine (PE); U-46,619 (thromboxane-prostanoid receptor agonist) and endothelin-1 (ET-1). NO activity (DAF-FM fluorescence) and ROS generation (tempo-9-AC fluorescence) were measured by fluorescence microscopy. Data are mean ±SEM. The MAs from RRM mice had diminished EDR (54 ±5 vs. 77±3%; P<0.01) and EDRF (13±5 vs. 27±4%; P<0.01) with reduced NO activity (0.18 ± 0.05 vs. 0.36± 0.04 ΔUnits; P<0.05), but unchanged EDHF (30±4 vs. 38±4%; NS). These vessels from RRM mice developed an EDCF (14±1 vs. 8±1%; P<0.05) and ACh-induced increased in ROS (0.17±0.03 vs. 0.06±0.02 ΔUnits; P<0.05). Contractile responses were enhanced to U-46,619 (107±4 vs. 87±6, P<0.05) and ET-1 (108±7 vs. 89±4, P<0.05), but not to PE (87±6 vs. 77±8%, NS). Conclusion: mice with RRM developed defective microvascular EDR, EDRF with reduced NO activity and enhanced new ACh-induced EDCF, contractilities to thromboxane and endothelin with increased ROS generation. These microvascular disturbances may contribute to the later development of thrombosis, vascular remodeling and dysfunction in patients with CKD. *D. Wang and C.Wang: Equal contribution

Microvascular Endothelial Dysfunction and Enhanced Thromboxane and Endothelial Contractility in Patients with HIV

The prevalence of cardiovascular disease is increased with human immunodeficiency virus (HIV) infection, but the mechanism is unclear. We hypothesized that HIV increases microvascular reactive oxygen species, thereby impairing endothelial function and enhancing contractility. Subcutaneous microarterioles were isolated from gluteal skin biopsies in premenopausal, African American, HIV positive women receiving effective anti-retroviral therapy, but without cardiovascular risk factors except for increased body mass index (n=10) and healthy matched controls (n=10). The arterioles were mounted on myographs, preconstricted and relaxed with acetylcholine for: endothelium-dependent relaxation, endothelium-dependent relaxation factor (nitric oxide synthase-dependent relaxation), endothelium-dependent hyperpolarizing factor (potassium-channel dependent relaxation) and endothelium-independent relaxation (nitroprusside). Contractions were tested to endothelium-dependent contracting factor (acetylcholine contraction with blocked relaxation); phenylephrine, U-46,619 and endothelin-1. Plasma L-arginine and asymmetric dimethylarginine were measured by high performance capillary electrophoresis. The micro-arterioles from HIV positive women had significantly (% change in tension; P<0.05) reduced acetylcholine relaxation (-51 ± 6 vs. -78 ± 3%), endothelium-dependent relaxation factor (-28 ± 4 vs. -39 ± 3%), endothelium-dependent hyperpolarizing factor (-17 ± 4 vs. -37 ± 4%) and decreased nitric oxide activity (0.16 ± 0.03 vs. 0.70 ± 0.16 Δ unit) but unchanged nitroprusside relaxation. They had significantly enhanced endothelium-dependent contracting factor (+21 ± 6 vs. +7 ± 2%) and contractions to U-46,619 (+164 ± 10 vs. +117 ± 11%) and endothelin-1(+151 ± 12 vs. +97 ± 9%), but not to phenylephrine. There was enhanced reactive oxygen species with acetylcholine (0.11 ± 0.02 vs. 0.05 ± 0.01 Δ unit; P<0.05) and endothelin-1 (0.31 ± 0.06 vs. 0.10 ± 0.02 Δ unit; P<0.05). Plasma L-arginine: assymetric dimethyl arginine rates was reduced (173 ± 12 vs. 231 ± 6 μmol·μmol-1, P<0.05). Premenopausal HIV positive womenhad microvascular oxidative stress with severe endothelial dysfunction and reduced nitric oxide and arginine: assymetric dimethylarginine ratio but enhanced endothelial, thromboxane and endothelin contractions. These microvascular changes may herald later cardiovascular disease.

Impaired Endothelial Function in Ovariectomised Rats: Effects of Tempol

Menopause causes endothelial dysfunction. We investigated the hypothesis that reactive oxygen species (ROS) mediate the adverse effects of ovariectomy (OVX) on microvessels. Mesenteric arterioles (MAs) were isolated from rats 4–6 weeks after sham‐operation (S) or OVX alone, S or OVX rats were given vehicle or tempol (2 mmol · l−1) throughout (n=6/group) to measure endothelium derived relaxation factor (EDRF) and endothelium dependent contracting factor (EDCF) using a wire myograph. NO and ROS were quantitated by DAF‐FM and tempo‐9AC‐fluorescence using RatioMasterTM. OVX rats had increased mean blood pressure (MBP) (114±7 vs 88±6.5 mmHg, P&lt;0.05). Their MAs had increased medium:lumen ratio (4.44±0.38 vs 1.54±0.29, P&lt;0.01) and EDRF (20±4 vs 41±4%, P&lt;0.001) and decreased NO activity (0.37±0.1 vs 0.7±0.1 Units, P&lt;0.01). However, OVX rats developed a new EDCF response (20±6 vs 5±5%, P&lt;0.05) accompanied by increased ROS activity (0.22±0.09 vs 0.10±0.01 units; P&lt;0.01). Tempol prevented significant changes in BP, vascular remodeling, EDRF and NO activity, and prevented any EDCF or ROS responses in OVX rats. In conclusion, OVX causes hypertension and microvascular remodeling and redirects endothelial responses from relaxation to contraction accompanied by reduced vascular NO and enhanced ROS. All of these effects are dependent on ROS and could, therefore, be targeted with effective antioxidant therapy.

Beneficial Vascular Effect of A Non‐selective PPAR Activator In Aorta of Spontaneously Hypertensive Rats

Wy14643 is a potent activator of peroxisome proliferator‐activated receptor (PPAR) α and γ. The present study aimed to determine whether or not Wy14643 improved endothelial dysfunction in hypertension, and if so, the mechanism involved. Isometric tension in isolated thoracic aortic rings of spontaneously hypertensive rats was recorded. Wy14643 caused greater relaxations than fenofibrate (PPARα activator) or rosiglitazone (PPARγ activator). L‐NAME (nitric oxide synthase inhibitor) and ODQ (soluble guanylyl cyclase inhibitor) alone and in combination inhibited these relaxations to the same extent. Compound C (AMP‐activated protein kinase (AMPK) inhibitor) reduced Wy14643‐induced relaxations to the same extent as L‐NAME. Endothelium‐dependent contractions evoked by acetylcholine in the presence of L‐NAME were reduced by Wy14643, fenofibrate but not rosiglitazone. Moreover, Wy14643 and fenofibrate inhibited acetylcholine‐induced prostanoids release to the same extent. Our data suggests that the three PPAR activators induce nitric oxide‐mediated relaxation through activation of AMPK. This relaxing effect is more prominent with Wy14643. Together with the ability to reduce the release of endothelium‐dependent contracting factor, it appears that Wy14643 produces better protection against vascular diseases in spontaneous hypertension.

Role of TNF-α in vascular dysfunction

Healthy vascular function is primarily regulated by several factors including EDRF (endothelium-dependent relaxing factor), EDCF (endothelium-dependent contracting factor) and EDHF (endothelium-dependent hyperpolarizing factor). Vascular dysfunction or injury induced by aging, smoking, inflammation, trauma, hyperlipidaemia and hyperglycaemia are among a myriad of risk factors that may contribute to the pathogenesis of many cardiovascular diseases, such as hypertension, diabetes and atherosclerosis. However, the exact mechanisms underlying the impaired vascular activity remain unresolved and there is no current scientific consensus. Accumulating evidence suggests that the inflammatory cytokine TNF (tumour necrosis factor)-α plays a pivotal role in the disruption of macrovascular and microvascular circulation both in vivo and in vitro. AGEs (advanced glycation end-products)/RAGE (receptor for AGEs), LOX-1 [lectin-like oxidized low-density lipoprotein receptor-1) and NF-κB (nuclear factor κB) signalling play key roles in TNF-α expression through an increase in circulating and/or local vascular TNF-α production. The increase in TNF-α expression induces the production of ROS (reactive oxygen species), resulting in endothelial dysfunction in many pathophysiological conditions. Lipid metabolism, dietary supplements and physical activity affect TNF-α expression. The interaction between TNF-α and stem cells is also important in terms of vascular repair or regeneration. Careful scrutiny of these factors may help elucidate the mechanisms that induce vascular dysfunction. The focus of the present review is to summarize recent evidence showing the role of TNF-α in vascular dysfunction in cardiovascular disease. We believe these findings may prompt new directions for targeting inflammation in future therapies.

Endothelium‐dependent contractions: when a good guy turns bad!

Endothelial cells can induce contractions of the underlying vascular smooth muscle by generating vasoconstrictor prostanoids (endothelium-dependent contracting factor; EDCF). The endothelial COX-1 isoform of cyclooxygenase appears to play the dominant role in the phenomenon. Its activation requires an increase in intracellular Ca(2+) concentration. The production of EDCF is inhibited acutely and chronically by nitric oxide (NO), and possibly by endothelium-dependent hyperpolarizing factor (EDHF). The main prostanoids involved in endothelium-dependent contractions appear to be endoperoxides (PGH(2)) and prostacyclin, which activate thromboxane-prostanoid (TP) receptors of the vascular smooth muscle cells. Oxygen-derived free radicals can facilitate the production and/or the action of EDCF. Endothelium-dependent contractions are exacerbated by ageing, obesity, hypertension and diabetes, and thus are likely to contribute to the endothelial dysfunction observed in older people and in essential hypertensive patients.

Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery.

In addition to endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium-derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H2O2) in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh). ACh (10(-6), 10(-5), and 10(-4) M) induced a transient contraction of rat renal artery with intact endothelium in a concentration-related manner, but not in the artery with endothelium removed. In phenylephrine-precontracted renal arteries, ACh induced an endothelium-dependent relaxation response at lower concentrations (10(-8)-10(-6) M), and a relaxation followed by a contraction at higher concentrations (10(-5) M). Inhibition of nitric oxide synthase by N(omega)-nitro-L-arginine (10(-4) M) enhanced the EDC to ACh. Catalase (1000 U ml(-1)) reduced the EDC to ACh. H2O2 (10(-6), 10(-5), and 10(-4) M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium-independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh-induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17-octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H2 receptors (SQ 29548) and thromboxane A2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H2O2. In isolated endothelial cells, ACh (10(-5) M) induced a transient H2O2 production detected with a fluorescence dye sensitive to H2O2 (2',7'-dichlorofluorescein diacetate). The peak concentration of H2O2 was 5.1 x 10(-4) M at 3 min and was prevented by catalase. Taken together, these results show that ACh triggers H2O2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H2O2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H2O2 is most likely the EDCF in rat renal artery in response to ACh stimulation.

Age-related changes in vascular responses.

Aging causes changes in the structure and function of the vessels that leads to an increase in the incidence of certain cardiovascular diseases, such as hypertension, coronary artery disease, heart failure, and postural hypotension with enhancement of both morbidity and mortality. When aging is associated with hypertension, these changes are increased. Aging alters endothelial cells, and so the vascular tone regulation, reducing the endothelium-dependent relaxations, probably by a decrease in endothelial synthesis or release of nitric oxide. In addition, endothelium-independent relaxations are essentially unaltered, those elicited by beta-adrenoceptor agonists being usually reduced. Aging scarcely modifies the contractions induced by different agents, such as 5-hydroxytryptamine, histamine, high potassium, and angiotensin, whereas reduces those elicited by noradrenaline or endothelin. Vascular Ca(2+) homeostasis appears to be altered in aging. The extracellular Ca(2+) dependence of contractile responses elicited by agonists is enhanced, which explains the increased sensitivity to Ca(2+) antagonists in elderly. Finally, Na(+) pump activity, that controls cellular ionic homeostasis, seems to be reduced in aging. The contractions elicited by Na(+) pump inhibition with ouabain are negatively modulated by the release of a diffusible endothelial factor, an effect lost in aging, being replaced by an endothelium-dependent contracting factor that facilitates ouabain responses.

Effect of ageing and hypertension on endothelial modulation of ouabain-induced contraction and sodium pump activity in the rat aorta.

To investigate the endothelial modulation of the rat thoracic aorta sodium-potassium ATPase activity and its possible alteration by ageing and hypertension. Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) aged 5 weeks, 3, 6, 12 and 18 months were anaesthetized. Their aortae were dissected and divided into cylindrical segments and vasomotor responses obtained in segments with or without endothelium were recorded on a polygraph. Endothelium removal increased ouabain responses in young (aged 3 and 6 months) WKY rat aortic segments, but reduced those obtained in old (aged 18 months) WKY rat segments and in segments from SHR aged 3, 6 and 12 months. An enhancement of contractions in response to ouabain with age was observed in both strains, but it occurred at earlier ages in vessels from SHR. At ages 3 and 6 months, responses to ouabain were higher in intact vessels from SHR than they were in those from WKY, but were lower in endothelium-denuded vessels. In aortic segments from 6-month-old WKY rats and SHR, ouabain reduced or abolished potassium-induced relaxations in segments with or without endothelium, respectively. In vessels from SHR and WKY rats aged 18 months, ouabain abolished potassium-induced relaxations both in intact and in endothelium-denuded vessels. Endothelium of WKY rat aorta might release a factor in response to ouabain that modulates its contraction negatively. Endothelium of SHR and old WKY rat aorta releases a contracting factor that modulates ouabain contractions positively. Thus, the inhibitory effect of endothelium on contractions induced by ouabain might be lost as a consequence both of hypertension and of age, being replaced by an endothelium-dependent contracting factor that facilitates ouabain responses.

Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta. PGH2 does not contribute to impaired endothelium-dependent relaxation.

We examined whether prostaglandin (PG) H2, as an endothelium-dependent contracting factor, or the disturbed production of endothelium-derived relaxing factor, impairs endothelium-dependent relaxation and whether long-term inhibition of nitric oxide (NO) synthesis aggravates atherosclerosis in hypercholesterolemic rabbits. Male New Zealand White rabbits were fed one of the following diets: (1) standard chow; (2) 2% cholesterol-supplemented chow; (3) standard chow with 80 micrograms/mL N omega-nitro-L-arginine methylester (L-NAME), an NO synthetase inhibitor, in their drinking water; or (4) 2% cholesterol-supplemented chow with 80 or 160 micrograms/mL L-NAME in their drinking water. The rabbits were fed these diets for 8 or 12 weeks. Then aortic rings were obtained, and changes in isometric tension were recorded. Intimal atherosclerotic areas of the thoracic aortas were subsequently measured by planimetry. The cholesterol-supplemented diet significantly impaired endothelium-dependent aortic relaxation to acetylcholine. Pretreatment with the thromboxane A2/PGH2 receptor antagonist ONO-3708 did not reverse this impaired response. Vessels from both normocholesterolemic and hypercholesterolemic rabbits given L-NAME showed more impaired endothelium-dependent relaxation than those from their dietary counterparts not given L-NAME. Morphometric analysis revealed marked enlargement of intimal atherosclerotic areas in aortas from L-NAME-treated hypercholesterolemic rabbits compared with those from untreated hypercholesterolemic rabbits. These findings suggest that PGH2 does not contribute to impaired endothelium-dependent relaxation and that long-term administration of L-NAME promotes atherosclerosis by inhibition of NO synthesis in the hypercholesterolemic rabbit thoracic aorta.

Endothelium-dependent contractions in rabbit pulmonary artery are mediated by thromboxane A2.

This study was designed to characterize the endothelium-dependent contracting factor (EDCF) released by arachidonic acid (AA) and methacholine (MeCH) in the rabbit pulmonary artery. AA and MeCH contract the rabbit pulmonary artery; however, the effects of both are blocked by denuding the vessels and by administration of indomethacin (a cyclooxygenase inhibitor), dazoxiben (a thromboxane [TX] synthase inhibitor), and SQ29548 (a TXA2/prostaglandin [PG] H2 receptor antagonist). When segments of rabbit pulmonary artery were incubated with [14C]AA and the [14C] metabolites were resolved by reverse-phase high-performance liquid chromatography (HPLC), radioactive products were observed that comigrated with 6-keto-PGF1 alpha and TXB2, the stable metabolites of prostacyclin and TXA2. The TXB2 radioactive peak was rechromatographed on normal-phase HPLC and again migrated with TXB2. Finally, the structures of derivatized [14C]6-keto-PGF1 alpha and [14C]TXB2 peaks were confirmed by gas chromatography/mass spectrometry. The synthesis of [14C]6-keto-PGF1 alpha and [14C]TXB2 was inhibited by removal of the endothelium and by indomethacin. Dazoxiben inhibited the synthesis of [14C]TXB2 but not [14C]6-keto-PGF1 alpha. Using specific radioimmunoassays, AA and MeCH stimulated 6-keto-PGF1 alpha and TXB2 release. Indomethacin blocked the production of both 6-keto-PGF1 alpha and TXB2, whereas dazoxiben only blocked TXB2. In a superfusion/bioassay system, AA stimulated an endothelium-intact donor vessel to release a labile substance that contracted an indomethacin-treated endothelium-denuded recipient vessel. The EDCF released by AA had an approximate half-life of 30 seconds. Cultured rabbit pulmonary arterial endothelial cells synthesized 6-keto-PGF1 alpha but not TXB2. Immunohistochemical studies indicated the presence of cyclooxygenase, but not TX synthase, in pulmonary artery endothelial cells. TXA2 appears to be the EDCF released by AA and MeCH in rabbit pulmonary artery; however, TXA2 is not produced by endothelial cells but may arise from cells that adhere to the luminal surfaces, such as platelets or macrophages.

Endothelium-Dependent Contractions

The endothelial cells help to control the tone of the underlying vascular smooth muscle by releasing vasoactive factors. In physiological circumstances, the release of relaxing factors (nitric oxide and endothelium-derived hyperpolarizing factor) appears to predominate. However, in certain blood vessels (peripheral veins and large cerebral arteries), the normal endothelium has the propensity to release vasoconstrictor substances, among which are superoxide anion and thromboxane A2; the release of these endothelium-derived vasoconstrictors may contribute to the autoregulatory processes. In most blood vessels, anoxic conditions initiate the release of an unidentified endothelium-dependent contracting factor. Cultured endothelial cells, and blood vessels maintained under culture conditions for prolonged periods of time, release the vasoconstrictor peptide endothelin. A characteristic of vascular diseases is that the ability of the endothelial cells to release relaxing factor(s) is reduced, while the generation of contracting factor is maintained or enhanced.