Xiaoqiang Yao

Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure

The kidney not only regulates fluid and electrolyte balance but also functions as an endocrine organ. For instance, it is the major source of circulating erythropoietin and renin. Despite currently available therapies, there is a marked increase in cardiovascular morbidity and mortality among patients suffering from end-stage renal disease. We hypothesized that the current understanding of the endocrine function of the kidney was incomplete and that the organ might secrete additional proteins with important biological roles. Here we report the identification of a novel flavin adenine dinucleotide-dependent amine oxidase (renalase) that is secreted into the blood by the kidney and metabolizes catecholamines in vitro (renalase metabolizes dopamine most efficiently, followed by epinephrine, and then norepinephrine). In humans, renalase gene expression is highest in the kidney but is also detectable in the heart, skeletal muscle, and the small intestine. The plasma concentration of renalase is markedly reduced in patients with end-stage renal disease, as compared with healthy subjects. Renalase infusion in rats caused a decrease in cardiac contractility, heart rate, and blood pressure and prevented a compensatory increase in peripheral vascular tone. These results identify renalase as what we believe to be a novel amine oxidase that is secreted by the kidney, circulates in blood, and modulates cardiac function and systemic blood pressure.

Exercise, Vascular Wall and Cardiovascular Diseases An Update (Part 1)

Cardiovascular disease (CVD) remains the leading cause of morbidity and premature mortality in both women and men in most industrialized countries, and has for some time also established a prominent role in developing nations. In fact, obesity, diabetes mellitus and hypertension are now commonplace even in children and youths. Regular exercise is rapidly gaining widespread advocacy as a preventative measure in schools, medical circles and in the popular media. There is overwhelming evidence garnered from a number of sources, including epidemiological, prospective cohort and intervention studies, suggesting that CVD is largely a disease associated with physical inactivity. A rapidly advancing body of human and animal data confirms an important beneficial role for exercise in the prevention and treatment of CVD.In Part 1 of this review we discuss the impact of exercise on CVD, and we highlight the effects of exercise on (i) endothelial function by regulation of endothelial genes mediating oxidative metabolism, inflammation, apoptosis, cellular growth and proliferation, increased superoxide dismutase (SOD)-1, down-regulation of p67phox, changes in intracellular calcium level, increased vascular endothelial nitric oxide synthase (eNOS), expression and eNOS Ser-1177 phosphorylation; (ii) vascular smooth muscle function by either an increased affinity of the Ca2+ extrusion mechanism or an augmented Ca2+ buffering system by the superficial sarcoplasmic reticulum to increase Ca2+ sequestration, increase in K+ channel activity and/or expression, and increase in L-type Ca2+ current density; (iii) antioxidant systems by elevation of Mn-SOD, Cu/Zn-SOD and catalase, increases in glutathione peroxidase activity and activation of vascular nicotinamide adenine dinucleotide phosphate [(NAD(P)H] oxidase and p22phox expression; (iv) heat shock protein (HSP) expression by stimulating HSP70 expression in myocardium, skeletal muscle and even in human leucocytes, probably through heat shock transcription factor 1 activity; (v) inflammation by reducing serum inflammatory cytokines such as high-sensitivity C-reactive protein (hCRP), interleukin (IL)-6, IL-18 and tumour necrosis factor-α and by regulating Toll-like receptor 4 pathway.Exercise also alters vascular remodelling, which involves two forms of vessel growth including angiogenesis and arteriogenesis. Angiogenesis refers to the formation of new capillary networks. Arteriogenesis refers to the growth of pre-existent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. Another aim of this review is to focus on exercise-related cardiovascular protection against CVD and associated risk factors such as aging, coronary heart disease, hypertension, heart failure, diabetes mellitus and peripheral arterial diseases mediated by vascular remodelling. Lastly, this review examines the benefits of exercise in mitigating pre-eclampsia during pregnancy by mechanisms that include improved blood flow, reduced blood pressure, enhanced placental growth and vascularity, increased activity of antioxidant enzymes, reduced oxidative stress and restored vascular endothelial dysfunction.

Reactive Oxygen Species in Vascular Wall

Reactive oxygen species (ROS) contribute to the pathogenesis of cardiovascular diseases including hypertension, atherosclerosis, cardiac hypertrophy, heart failure and diabetes mellitus. Oxidative stress is resulted from excessive generation of ROS that outstrips the antioxidant system. Various agonists, pathological conditions and therapeutic interventions lead to modulated expression and function of oxidant and antioxidant enzymes, including NAD(P)H oxidase, endothelial nitric oxide synthase, xanthine oxidase, myeloperoxidase, superoxide dismutases, catalase and glutathione peroxidase. ROS formed in vascular wall target a wide range of signaling molecules and cellular pathways in both endothelium and vascular smooth muscle, such as transcription factors, protein tyrosine phosphatase, protein tyrosine kinase, mitogen-activated protein kinase, Ca(2+)-transporting system and protein modification. ROS also have distinct physiological and pathophysiological impacts on vascular cells. ROS contribute to vascular dysfunction and remodeling through oxidative damage by (1) reducing the bioavailability of NO, (2) impairing endothelium-dependent vasodilatation and endothelial cell growth, (3) causing apoptosis or anoikis, (4) stimulating endothelial cell migration, and (5) activating adhesion molecules and inflammatory reaction, leading to endothelial dysfunction, an initial episode progressing toward hypertension and atherosclerosis. Cellular events underlying these processes involve changes in vascular smooth muscle cell growth, apoptosis/anoikis, cell migration, inflammation, and vasoconstriction. The present communication focuses on the biology of ROS signaling in vascular cells, discusses how oxidative stress contributes to vascular damage, and the therapeutic strategies/biotic factors that can prevent or treat ROS-associated cardiovascular disorders.

BIOLOGICAL PROPERTIES OF BAICALEIN IN CARDIOVASCULAR SYSTEM

The dried roots of Scutellaria baicalensis (S. baicalensis) Georgi (common name: Huangqin in China) have been widely employed for many centuries in traditional Chinese herbal medicine as popular antibacterial and antiviral agents. They are effective against staphylococci, cholera, dysentery, pneumococci and influenza virus. Baicalein, one of the major flavonoids contained in the dried roots, possesses a multitude of pharmacological activities. The glycoside of baicalein, baicalin is a potent anti-inflammatory and anti-tumor agent. This review describes the biological properties of baicalein (Table 1), which are associated with the prevention and treatment of cardiovascular diseases. Baicalein is a potent free radical scavenger and xanthine oxidase inhibitor, thus improving endothelial function and conferring cardiovascular protective actions against oxidative stress-induced cell injury. Baicalein lowers blood pressure in renin-dependent hypertension and the in vivo hypotensive effect may be partly attributed to its inhibition of lipoxygenase, resulting in reduced biosynthesis and release of arachidonic acid-derived vasoconstrictor products. On the other hand, baicalein enhances vasoconstricting sensitivity to receptor-dependent agonists such as noradrenaline, phenylephrine, serotonin, U46619 and vasopressin in isolated rat arteries. The in vitro effect is likely caused by inhibition of an endothelial nitric oxide-dependent mechanism. The anti-thrombotic, anti-proliferative and anti-mitogenic effects of the roots of S. baicalensis and baicalein are also reported. Baicalein inhibits thrombin-induced production of plasminogen activator inhibitor-1, and interleukin-1beta- and tumor necrosis factor-alpha-induced adhesion molecule expression in cultured human umbilical vein endothelial cells. The pharmacological findings have highlighted the therapeutic potentials of using plant-derived baicalein and its analogs for the treatment of arteriosclerosis and hypertension.

Cyclooxygenase-2–Derived Prostaglandin F2α Mediates Endothelium-Dependent Contractions in the Aortae of Hamsters With Increased Impact During Aging

Hypertension and vascular dysfunction result in the increased release of endothelium-derived contracting factors (EDCFs), whose identity is poorly defined. We tested the hypothesis that endothelial cyclooxygenase (COX)-2 can generate EDCFs and identified the possible EDCF candidate. Changes in isometric tension of aortae of young and aged hamsters were recorded on myograph. Real-time changes in intracellular calcium concentrations ([Ca2+]i) in native aortic endothelial cells were measured by imaging. Endothelium-dependent contractions were triggered by acetylcholine (ACh) after inhibition of nitric oxide production and they were abolished by COX-2 but not COX-1 inhibitors or by thromboxane–prostanoid receptor antagonists. 2-Aminoethoxydiphenyl borate (cation channel blocker) eliminated endothelium-dependent contractions and ACh-stimulated rises in endothelial cell [Ca2+]i. RT-PCR and Western blotting showed COX-2 expression mainly in the endothelium. Enzyme immunoassay and high-performance liquid chromatography-coupled mass spectrometry showed release of prostaglandin (PG)F2&agr; and prostacyclin (PGI2) increased by ACh; only PGF2&agr; caused contraction at relevant concentrations. COX-2 expression, ACh-stimulated contractions, and vascular sensitivity to PGF2&agr; were augmented in aortae from aged hamsters. Human renal arteries also showed thromboxane–prostanoid receptor–mediated ACh- or PGF2&agr;-induced contractions and COX-2–dependent release of PGF2&agr;. The present study demonstrates that PGF2&agr;, derived from COX-2, which is localized primarily in the endothelium, is the most likely EDCF underlying endothelium-dependent, thromboxane–prostanoid receptor–mediated contractions to ACh in hamster aortae. These contractions involved increases in endothelial cell [Ca2+]i. The results support a critical role of COX-2 in endothelium-dependent contractions in this species with an increased importance during aging and, possibly, a similar relevance in humans.

Dipeptidyl Peptidase 4 Inhibitor Sitagliptin Protects Endothelial Function in Hypertension Through a Glucagon–Like Peptide 1–Dependent Mechanism

Sitagliptin, a selective dipeptidyl peptidase 4 inhibitor, inhibits the inactivation and degradation of glucagon like peptide 1 (GLP-1), which is used for the treatment of type 2 diabetes mellitus. However, little is known about the role of GLP-1 in hypertension. This study investigated whether the activation of GLP-1 signaling protects endothelial function in hypertension. Two-week sitagliptin treatment (10 mg/kg per day, oral gavage) improved endothelium-dependent relaxation in renal arteries, restored renal blood flow, and reduced systolic blood pressure in spontaneously hypertensive rats. In vivo sitagliptin treatment elevated GLP-1 and GLP-1 receptor expressions, increased cAMP level, and subsequently activated protein kinase A, liver kinase B1, AMP-activated protein kinase-&agr; and endothelial NO synthase in spontaneously hypertensive rat renal arteries. Inhibition of GLP-1 receptor, adenylyl cyclase, protein kinase A, AMP-activated protein kinase-&agr;, or NO synthase reversed the protective effects of sitagliptin. We also demonstrate that GLP-1 receptor agonist exendin 4 in vitro treatment had similar vasoprotective effects in spontaneously hypertensive rat renal arteries and increased NO production in spontaneously hypertensive rat aortic endothelial cells. Studies using transient expressions of wild-type and dominant-negative AMP-activated protein kinase-&agr;2 support the critical role of AMP-activated protein kinase-&agr; in mediating the effect of GLP-1 in endothelial cells. Ex vivo exendin 4 treatment also improved endothelial function of renal arteries from hypertensive patients. Our results elucidate that upregulation of GLP-1 and related agents improve endothelial function in hypertension by restoring NO bioavailability, suggesting that GLP-1 signaling could be a therapeutic target in hypertension-related vascular events.

Organelle pH in the Arabidopsis endomembrane system.

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein (GFP) (smGFP) with the pH-sensing capability of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin (plant-solubility-modified ecliptic pHluorin) gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin (plant-solubility-modified ratiomatric pHluorin), a dual-excitation sensor, allowing for precise measurements. Compartment-specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar (pH 7.3 and 7.2), while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum (ER) to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network (TGN) and multivesicular body (MVB) is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H(+)-ATPase (V-ATPase) with concanamycin A (ConcA) caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.

Store-operated Calcium Entry in Vascular Endothelial Cells Is Inhibited by cGMP via a Protein Kinase G-dependent Mechanism

Store-operated Ca2+ entry in vascular endothelial cells not only serves to refill the intracellular Ca2+ stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca2+ entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca2+-ATPase, were used to induce store-operated Ca2+ entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca2+ entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 μm) or H-8 (10 μm), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca2+ entry. Addition ofS-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca2+ influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca2+-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca2+ entry in vascular endothelial cells.

Store‐operated calcium entry in vascular smooth muscle

In non‐excitable cells, activation of G‐protein‐coupled phospholipase C (PLC)‐linked receptors causes the release of Ca2+ from intracellular stores, which is followed by transmembrane Ca2+ entry. This Ca2+ entry underlies a small and sustained phase of the cellular [Ca2+]i increases and is important for several cellular functions including gene expression, secretion and cell proliferation. This form of transmembrane Ca2+ entry is supported by agonist‐activated Ca2+‐permeable ion channels that are activated by store depletion and is referred to as store‐operated Ca2+ entry (SOCE) and represents a major pathway for agonist‐induced Ca2+ entry. In excitable cells such as smooth muscle cells, Ca2+ entry mechanisms responsible for sustained cellular activation are normally considered to be mediated via either voltage‐operated or receptor‐operated Ca2+ channels. Although SOCE occurs following agonist activation of smooth muscle, this was thought to be more important in replenishing Ca2+ stores rather than acting as a source of activator Ca2+ for the contractile process. This review summarizes our current knowledge of SOCE as a regulator of vascular smooth muscle tone and discusses its possible role in the cardiovascular function and disease. We propose a possible hypothesis for its activation and suggest that SOCE may represent a novel target for pharmacological therapeutic intervention.

Uncoupling Protein-2 Protects Endothelial Function in Diet-Induced Obese Mice

Rationale: Previous studies indicate uncoupling protein-2 (UCP2) as an antioxidant defense against endothelial dysfunction in hypertension. UCP2 also regulates insulin secretion and action. However, the role of UCP2 in endothelial dysfunction associated with diabetes and obesity is unclear. Objective: UCP2 protects against endothelial dysfunction induced by high-fat diet through inhibition of reactive oxygen species (ROS) production, and subsequent increase of nitric oxide bioavailability. Methods and Results: Endothelium-dependent relaxation (EDR) in aortae and mesenteric arteries in response to acetylcholine was measured in wire myograph. Flow-mediated vasodilatation in 2nd-order mesenteric arteries was measured in pressure myograph. ROS production is measured by CM-H2DCFDA and DHE fluorescence. High-glucose exposure reduced EDR in mouse aortae, which was exaggerated in UCP2 knockout (KO) mice, whereas UCP2 overexpression by adenoviral infection (AdUCP2) restored the impaired EDR. Impairment of EDR and flow-mediated vasodilatation in aortae and mesenteric arteries from high-fat diet-induced obese mice (DIO) was exaggerated in UCP2KO DIO mice compared with wild-type DIO littermates, whereas AdUCP2 i.v. injection restored both EDR and flow-mediated vasodilatation in DIO mice. Improved EDR in mesenteric arteries was inhibited by nitric oxide synthase inhibitor. UCP2 overexpression also inhibited intracellular ROS production in the en face endothelium of aorta and mesenteric artery of DIO mice, whereas UCP2 deficiency enhanced ROS production. Conclusions: UCP2 preserves endothelial function through increasing nitric oxide bioavailability secondary to the inhibition of ROS production in the endothelium of obese diabetic mice.