Jesus Ramon Castillo-Hernandez

Two dissimilar AT1 agonists distinctively activate AT1 receptors located on the luminal membrane of coronary endothelium

Diverse intracoronary agonists cause cardiac effects while acting on coronary endothelial luminal membrane (CELM) receptor. Our data show: a) the presence of AT(1)R in isolated CELM and in all cardiac cell types and b) sustained intracoronary infusions of Ang II-POL, a large sized molecule (approximately 15,000 kDa) confined to the vessel lumen that can only act on CELMs AT(1)R or Ang II (approximately 1 kDa); both exert the same maximum positive inotropic (PIE) and coronary constriction (CPP). The effects of these two agonists are blocked by Losartan and by Sar-POL; a large size antagonist (approximately 15,000 kDa) that acts only on CELM. Ang II effects are transient due to desensitization and cause tachyphylaxis to Ang II and toward Ang II-POL suggesting that both Ang II and Ang II-POL act on the same receptor group. In contrast, Ang II-POL effects are sustained and do not cause tachyphylaxis. The results show that intravascular Ang II and Ang II-POL act differentially by an unknown mechanism on CELMs AT(1)R and suggest that intravascular Ang II and Ang II-POL cause PIE and CCP by activation limited to CELMs AT(1)R through an unknown mechanism that is space-confined to the CELMs AT(1)R.

Luminal endothelial lectins with affinity for N-acetylglucosamine determine flow-induced cardiac and vascular paracrine-dependent responses.

Coronary blood flow applied to the endothelial lumen modulates parenchymal functions via paracrine effectors, but the mechanism of flow sensation is unknown. We and others have demonstrated that coronary endothelial luminal membrane (CELM) oligosaccharides and lectins are involved in flow detection, and we proposed that cardiac effects of coronary flow result from a reversible flow-modulated lectin-oligosaccharide interaction. Recently, glycosylated and amiloride-sensitive Na(+)/Ca(++) channels (ENaCs) have been proposed to be involved in the flow-induced endothelial responses. Because N-acetylglucosamine (GlcNac) is one of the main components of glycocalyx oligosaccharides (i.e., hyaluronan [-4GlcUAbeta1-3GlcNAcbeta1-](n)), the aim of this article is to isolate and define CELM GlcNac-binding lectins and determine their role in cardiac and vascular flow-induced effects. For this purpose, we synthesized a 460-kDa GlcNac polymer (GlcNac-Pol) with high affinity toward GlcNac-recognizing lectins. In the heart, intracoronary administration of GlcNac-Pol upon binding to CELM diminishes the flow-dependent positive inotropic and dromotropic effects. Furthermore, GlcNac-Pol was used as an affinity probe to isolate CELM GlcNac-Pol-recognizing lectins and at least 35 individual lectinic peptides were identified, one of them the beta-ENaC channel. Some of these lectins could participate in flow sensing and in GlcNac-Pol-induced effects. We also adopted a flow-responsive and well-accepted model of endothelial-parenchymal paracrine interaction: isolated blood vessels perfused at controlled flow rates. We established that flow-induced vasodilatation (FIV) is blocked by endothelial luminal membrane (ELM) bound GlcNac-Pol, nitro-l-arginine methyl ester and indomethacin, amiloride, and hyaluronidase. The effect of hyaluronidase was reversed by infusion of soluble hyaluronan. These results indicate that GlcNac-Pol inhibits FIV by competing and displacing intrinsic hyaluronan bound to a lectinic structure such as the amiloride-sensitive ENaC. Nitric oxide and prostaglandins are the putative paracrine mediators of FIV.

ENZYMATIC HYDROLYSIS OF LUMINAL CORONARY GLYCOSIDIC STRUCTURES UNCOVERS THEIR ROLE IN SENSING CORONARY FLOW

Endothelial luminal glycocalyx (ELG) is a multifunctional complex structure made off of a diversity of glycosilated proteins, and glycosaminoglycans (GAG). Coronary ELG may participate as a sensor of coronary flow (CF) to induce inotropic and dromotropic effects. In isolated perfused guinea pig heart we tested the role of glycosidic groups of glycans bound to proteins and GAG of the ELG on CF-induced inotropic and dromotropic effects. To study the role of saccharide related groups of certain glycans, they were removed by selective enzyme hydrolysis or bound to a selective plant lectin. CF-induced positive inotropic and positive dromotropic control curves were obtained and the effects of intracoronary infusion of enzyme or lectin determined. The analyzed groups were as follow: 1) Fucosidase enzyme and Ulex europeasus lectin; hydrolysis and binding respectively (H&Br) to alpha-linked fucosyl related groups. 2). Endoglycanase-H and Lycopersicon esculentum (H&Br to N-linked beta-1,3GlcNAc related groups). 3) O-glycanase and Arachis hypogea (H&Br to O-linked beta-Gal1, 3GalNac related groups). 4) Sialidase and Maackia amurensis (H&Br to neuraminic acid related groups). In treatments 1-3 both. lectin and corresponding enzyme, equally depressed CF-positive dromotropic effects without affecting positive inotropic effects. In treatment 4 both lectin and enzyme equally depressed CF-positive inotropic effects without dromotropic effects. The differential role of GAG hyaluran or heparan groups on CF-positive inotropism and positive dromotropism respectively was shown. Infusing hyaluranidase removed hyaluran that solely inhibited CF- inotropism while removal of heparan with heparinase solely inhibited CF-dromotropism. Only the effects of hyaluronidase were reversed infusing hyaluronidate. Our results indicate glycans of ELG are elements of complex multimolecular sensors of coronary flow.

The coronary endothelium behaves as a functional diffusion barrier for intravascular Angiotensin II.

Diverse intracoronary hormones cause their cardiac effects solely via activation of their coronary endothelial luminal membrane (CELM) receptors. To test this hypothesis for Ang II, we synthesized: a) two large polymers of Ang II (Ang II-POL) and Losartan (Los-POL) which act only in the CELMs AT1R because they cannot cross the endothelial barrier and b) biotin-labeled Ang II (Ang II-Biotin) and Ang II-POL-Biotin to be identified by microscopy in tissues. Sustained coronary perfusion of Ang II (potentially diffusible) or Ang II-POL caused a positive inotropic effect (PIE) and an increase in coronary perfusion pressure (CPP) of equal magnitude that were blocked by Losartan and Los-POL. However, Ang II effects, in contrast to Ang II-POL effects, were transient due to desensitization and resulted in tachyphylaxis to a second administration of Ang II or Ang II-POL. Furthermore, if Ang II and Ang II-POL acted differently on the same receptor; a competition of effects would be expected. This was demonstrated by infusing simultaneously a molar ratio of Ang II:Ang II-POL. As this molar ratio decreased, Ang II-induced desensitization and tachyphylaxis decreased. Intravascularly-administered Ang II-Biotin and Ang II-POL-Biotin remained bound and confined to the endothelium. Our results support the hypothesis and indicate intravascular Ang II, not by mass exchange with the interstitium, but by an action restricted to the CELMs AT1R, causes release of endothelial chemical messengers that exert physiological effects and modulate the effects and metabolism of paracrine Ang II. Endocrine Ang II controls and communicates with its paracrine counterparts solely through endothelial receptors.

Obesity is the main determinant of insulin resistance more than the circulating pro-inflammatory cytokines levels in rheumatoid arthritis patients

BACKGROUND Systemic blockade of TNF-α in Rheumatoid arthritis with insulin resistance seems to produce more improvement in insulin sensitivity in normal weight patients with Rheumatoid arthritis than in obese patients with Rheumatoid arthritis, suggesting that systemic-inflammation and obesity are independent risk factors for insulin resistance in Rheumatoid arthritis patients. OBJECTIVES To evaluate the insulin resistance in: normal weight patients with Rheumatoid arthritis, overweight patients with Rheumatoid arthritis, obese Rheumatoid arthritis patients, and matched control subjects with normal weight and obesity; and its association with major cytokines involved in the pathogenesis of the disease. METHODS Assessments included: body mass index, insulin resistance by Homeostasis Model Assessment, ELISA method, and enzymatic colorimetric assay. RESULTS Outstanding results from these studies include: (1) In Rheumatoid arthritis patients, insulin resistance was well correlated with body mass index, but not with levels of serum cytokines. In fact, levels of cytokines were similar in all Rheumatoid arthritis patients, regardless of being obese, overweight or normal weight (2) Insulin resistance was significantly higher in Rheumatoid arthritis with normal weight than in normal weight (3) No significant difference was observed between insulin resistances of Rheumatoid arthritis with obesity and obesity (4) As expected, levels of circulating cytokines were significantly higher in Rheumatoid arthritis patients than in obesity. CONCLUSIONS Obesity appears to be a dominant condition above inflammation to produce IR in RA patients. The dissociation of the inflammation and obesity components to produce IR suggests the need of an independent therapeutic strategy in obese patients with RA.