Role of myocardium and endothelium in coronary vascular smooth muscle responses to hypoxia

Hypoxia triggers a mechanism that induces vasodilation in the whole heart but not necessarily in isolated coronary arteries. We therefore studied the role of cardiomyocytes (CM), smooth muscle cells (SMC), and endothelial cells (EC) in coronary responses to hypoxia (PO(2) of 5-10 mmHg). In an attempt to determine the factor(s) released in response to hypoxia, we inhibited the contribution of adenosine, ATP-sensitive K(+) channels, prostaglandins, and nitric oxide. Isolated rat septal artery segments without (-T) and with a layer of cardiac tissue (+T) were mounted in a double wire myograph, and constriction was induced. Hypoxia induced a decrease in isometric force of 21% and 61% in -T and +T segments, respectively (P < 0.05). EC removal increased the relaxation to hypoxia in -T segments to 33% but had the same effect in +T segments (61%). Only one of the inhibitors, the adenosine antagonist in +T segments, partially affected the relaxation due to hypoxia. The role of adenosine is thus limited and other mechanisms have to contribute. We conclude that hypoxia induces a relaxation of SMC that is augmented by the presence of CM and blunted by the endothelium. A single mediator does not induce those effects.     

The endothelium: influencing vascular smooth muscle in many ways

The endothelium, although only a single layer of cells lining the vascular and lymphatic systems, contributes in multiple ways to vascular homeostasis. Subsequent to the 1980 report by Robert Furchgott and John Zawadzki, there has been a phenomenal increase in our knowledge concerning the signalling molecules and pathways that regulate endothelial - vascular smooth muscle communication. It is now recognised that the endothelium is not only an important source of nitric oxide (NO), but also numerous other signalling molecules, including the putative endothelium-derived hyperpolarizing factor (EDHF), prostacyclin (PGI(2)), and hydrogen peroxide (H(2)O(2)), which have both vasodilator and vasoconstrictor properties. In addition, the endothelium, either via transferred chemical mediators, such as NO and PGI(2), and (or) low-resistance electrical coupling through myoendothelial gap junctions, modulates flow-mediated vasodilatation as well as influencing mitogenic activity, platelet aggregation, and neutrophil adhesion. Disruption of endothelial function is an early indicator of the development of vascular disease, and thus an important area for further research and identification of potentially new therapeutic targets. This review focuses on the signalling pathways that regulate endothelial - vascular smooth muscle communication and the mechanisms that initiate endothelial dysfunction, particularly with respect to diabetic vascular disease.     

Antioxidant-independent ascorbate enhancement of catecholamine-induced contractions of vascular smooth muscle

Ascorbate reduces the oxidation rate of catecholamines and, by an independent mechanism, enhances rabbit aortic ring contractions initiated by catecholamines. The largest significantly different fractional increases in force produced by ascorbate enhancement of norepinephrine (NE), epinephrine, phenylpropanolamine (PPA), and ephedrine (Eph) are 5.5, 1.8, 1.6, and 1.3 times, respectively. In physiological salt solutions bubbled with 95% O(2) at 37 degrees C, NE, PPA, and Eph have oxidation rate constants of 1.24, 247, and 643 h, respectively. Ascorbate significantly enhances 100 nM NE contractions by at least twofold at all ascorbate concentrations >15 microM, including the entire physiological range of 40-100 microM. Ascorbate preloading and washout followed by NE exposure produces significantly greater contractions than NE without ascorbate preloading but significantly lower than NE simultaneously with ascorbate. Ascorbate does not enhance K(+)- or angiotensin II-induced contractions. Ascorbate enhancement of catecholamine contractions occurs in addition to the reduction in oxidation rate, because the increases in force occur faster than oxidation can occur, the increases occur with compounds that have negligible oxidation rates, and the increases occur when ascorbate and NE are not physically present together. These results are consistent with ascorbate acting on the adrenergic receptor. Ascorbate may play a role in shock and asthma treatments and potentiate the cardiovascular health consequences of PPA and Eph (Ephedra).     

Role of the endothelium in the development of the contractile reactions of vascular smooth muscle during decreased oxygenation

Noradrenaline-preactivated vascular smooth muscles (VSM) of the rat thoracic aorta showed two-phase reactions in response to decreased oxygenation: significant relaxation was preceded by transient constriction. When the endothelium was removed only VSM relaxation phase was retained, with no constriction observed. The data obtained suggest an endothelium-dependent nature of VSM constriction reaction to hypoxia, in contrast to endothelium-independent VSM relaxation. Intracellular calcium is also assumed to play an essential role in the formation of endothelium-dependent constriction VSM reaction to hypoxia.     

A viscoelastic model of mechanically induced and spontaneous contractions of vascular smooth muscle

A new viscoelastic model was developed for the mathematical characterisation of mechanically induced and intrinsic contractional responses of the vascular smooth muscle. To this end, the elastic and viscous analogue elements were supplemented with a new active element generating stress proportional to its momentary elongation. The four-element model consisting of an active element, a parallel viscous element and both series and parallel elastic elements predicted biphasic or damped oscillatory stress relaxation and creep responses which were similar to that found experimentally earlier. Above a certain exciting frequency the model exhibited dissipative and below energy producing behaviour, as indicated by the sign change of the hysteresis loop area. In the case of sinusoidal modulation of the stress generation parameter the model showed parametric resonance, which was regarded as a simulation of intrinsic oscillation of the smooth muscle.     

iNOS expression in vascular resident macrophages contributes to circulatory dysfunction of splanchnic vascular smooth muscle contractions in portal hypertensive rats

Portal hypertension, a major complication of cirrhosis, is caused by both increased portal blood flow due to arterial vasodilation and augmented intrahepatic vascular resistance due to sinusoidal constriction. In this study, we examined the possible involvement of resident macrophages in the tone regulation of splanchnic blood vessels using bile duct ligated (BDL) portal hypertensive rats and an in vitro organ culture method. In BDL cirrhosis, the number of ED2-positive resident macrophages increased by two- to fourfold in the vascular walls of the mesenteric artery and extrahepatic portal vein compared with those in sham-operated rats. Many ED1-positive monocytes were also recruited into this area. The expression of inducible nitric oxide (NO) synthase (iNOS) mRNA was increased in the vascular tissues isolated from BDL rats, and accordingly, nitrate/nitrite production was increased. Immunohistochemistry revealed that iNOS was largely expressed in ED1-positive and ED2-positive cells. We further analyzed the effect of iNOS expression on vascular smooth muscle contraction using an in vitro organ culture system. iNOS mRNA expression and nitrate production significantly increased in vascular tissues (without endothelium) incubated with 1 μg/ml lipopolysaccharide (LPS) for 6 h. Immunohistochemistry indicated that iNOS was largely expressed in ED2-positive resident macrophages. α-Adrenergic-stimulated contractility of the mesenteric artery was greatly suppressed by LPS treatment and was restored by N(G)-nitro-L-arginine methyl ester (NO synthase inhibitor); in contrast, portal vein contractility was largely unaffected by LPS. Sodium nitroprusside (NO donor) and 8-bromo-cGMP showed greater contractile inhibition in the mesenteric artery than in the portal vein with decreasing myosin light chain phosphorylation. In the presence of an α-adrenergic agonist, the mesenteric artery cytosolic Ca(2+) level was greatly reduced by sodium nitroprusside; however, the portal vein Ca(2+) level was largely unaffected. These results suggest that the induction of iNOS in monocytes/macrophages contributes to a hypercirculatory state in the cirrhosis model rat in which the imbalance of the responsiveness of visceral vascular walls to NO (mesenteric artery > portal vein) may account for the increased portal venous flow in portal hypertension.     

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca2(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM2 but not with 2 × 1 or 1 × 4 Ca × P(i) mM2. Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.     

Functional changes in vascular smooth muscle and endothelium of arteries during diabetes mellitus.

To investigate the influence of diabetes mellitus on the responsiveness of the vascular smooth muscle, the effects of various vasoactive agents on the reactivity of the vascular smooth muscle from diabetic animals have been undertaken, focusing on the functional changes in the endothelium, alpha-adrenoceptors, beta-adrenoceptors, voltage-dependent Ca(2+)-channels, receptor-operated Ca(2+)-channels, phosphatidylinositol turnover and potassium channels. Among the functional changes, it is a common phenomenon that decreases in acetylcholine-induced production of cyclic GMP are due to the attenuation of release of endothelium-derived relaxing factor through an impairment of endothelium; this observation was found in both rats and rabbits with diabetes mellitus. These functional changes in diabetes may be responsible for the vascular complications such as coronary heart disease, cerebrovascular disease, and an acceleration in atherosclerosis.     

Role of vascular smooth muscle cell in the inflammation of atherosclerosis

Atherosclerosis is a pathologic process occurring within the artery, in which many cell types, including T cell, macrophages, endothelial cells, and smooth muscle cells, interact, and cause chronic inflammation, in response to various inner- or outer-cellular stimuli. Atherosclerosis is characterized by a complex interaction of inflammation, lipid deposition, vascular smooth muscle cell proliferation, endothelial dysfunction, and extracellular matrix remodeling, which will result in the formation of an intimal plaque. Although the regulation and function of vascular smooth muscle cells are important in the progression of atherosclerosis, the roles of smooth muscle cells in regulating vascular inflammation are rarely focused upon, compared to those of endothelial cells or inflammatory cells. Therefore, in this review, we will discuss here how smooth muscle cells contribute or regulate the inflammatory reaction in the progression of atherosclerosis, especially in the context of the activation of various membrane receptors, and how they may regulate vascular inflammation. [BMB Reports 2014; 47(1): 1-7]     

Role of cGMP in relaxation of vascular and other smooth muscle

The hypothesis that the relaxant action of many drugs on vascular and other smooth muscle is mediated by increases in intracellular cGMP, the "cGMP hypothesis," is gaining wide acceptance. While much information supporting this idea can be found in the literature, there is also a significant amount of information indicating that an elevation in the tissue content of cGMP is by itself insufficient to cause smooth muscle relaxation. The literature is reviewed with reference to the criteria that need to be fulfilled to consider cGMP as the second messenger mediating relaxation of smooth muscle by a drug; i.e., activation of guanylate cyclase, elevation of tissue content of cGMP, potentiation by phosphodiesterase inhibitors, antagonism by inhibitors of cGMP synthesis, and production of relaxation by cGMP analogues. For each criterion, key observations supporting the hypothesis are considered, followed by examples of important observations not consistent with the hypothesis. It is concluded that in some smooth muscles, for example, rat myometrium and vas deferens, cGMP is not a mediator of drug-induced relaxation. In other smooth muscles, including vascular smooth muscle, cGMP appears to play an important role in the relaxation process; but current evidence suggests that other factors are also important and that the cGMP hypothesis may need to be modified.     

Role of protein kinase C in the control of vascular prostacyclin: Study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca⁺⁺ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Role of protein kinase C in the control of vascular prostacyclin: study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca++ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Role of asymmetric dimethylarginine in homocysteine-induced apoptosis of vascular smooth muscle cells

Homocysteine (Hcy) could induce apoptosis of vascular smooth muscle cells (VSMC). Asymmetric dimethylarginine (ADMA) has been thought as a novel risk factor for cardiovascular diseases. We hypothesized that ADMA mediates homocysteine-induced apoptosis of VSMC. In this experiment the level of ADMA in the medium measured by high-performance liquid chromatography (HPLC) was elevated when the apoptosis of T/G HA-VSMC was induced by Hcy which was detected by Hoechst33342 staining or flow cytometry (FCM) with Annecin V+Propidium Iodide (PI). Exogenous ADMA induced the apoptosis of VSMC. At the same time, ADMA elevated the level of intracellular reactive oxidative species (ROS) determined by fluorescent ROS detection kit. The activation of JNK and p38MAPK contributed to ADMA-induced apoptosis of VSMC. The present results suggest that endogenous ADMA is involved in apoptosis of VSMC induced by Hcy, and the effects of ADMA is related to elevation of intracellular ROS and activation of JNK/p38MAPK signaling pathways.     

Role of integrins in angiotensin II-induced proliferation of vascular smooth muscle cells

Angiotensin II (AII) binds to G protein-coupled receptor AT(1) and stimulates extracellular signal-regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix, which occurs via integrins. To study cross-talk between G protein-coupled receptor- and integrin-induced signaling, we hypothesized that integrins are involved in AII-induced proliferation of VSMC. Using Oligo GEArray and quantitative RT-PCR, we established that messages for α(1)-, α(5)-, α(V)-, and β(1)-integrins are predominant in VSMC. VSMC were cultured on plastic dishes or on plates coated with either extracellular matrix or poly-d-lysine (which promotes electrostatic cell attachment independent of integrins). AII significantly induced proliferation in VSMC grown on collagen I or fibronectin, and this effect was blocked by the ERK inhibitor PD-98059, suggesting that AII-induced proliferation requires ERK activity. VSMC grown on collagen I or on fibronectin demonstrated approximately three- and approximately sixfold increases in ERK phosphorylation after stimulation with 100 nM AII, respectively, whereas VSMC grown on poly-d-lysine demonstrated no significant ERK activation, supporting the importance of integrin-mediated adhesion. AII-induced ERK activation was reduced by >65% by synthetic peptides containing an RGD (arginine-glycine-aspartic acid) sequence that inhibit α(5)β(1)-integrin, and by ～60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α(1)β(1). Furthermore, neutralizing antibody against β(1)-integrin and silencing of α(1), α(5), and β(1) expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α(5)β(1) and α(1)β(1)) in AII-induced proliferation of VSMC.