Patrícia Alessandra Bersanetti

ACE Activity Is Modulated by Kinin B2 Receptor

Angiotensin-converting enzyme (ACE) is an ectoprotein able to modulate the activity of a plethora of compounds, among them angiotensin I and bradykinin. Despite several decades of research, new aspects of the mechanism of action of ACE have been elucidated, expanding our understanding of its role not only in cardiovascular regulation but also in different areas. Recent findings have ascribed an important role for ACE/kinin B2 receptor heterodimerization in the pharmacological properties of the receptor. In this work, we tested the hypothesis that this interaction also affects ACE enzymatic activity. ACE catalytic activity was analyzed in Chinese hamster ovary cell monolayers coexpressing the somatic form of the enzyme and the receptor coding region using as substrate the fluorescence resonance energy transfer peptide Abz-FRK(Dnp)P-OH. Results show that the coexpression of the kinin B2 receptor leads to an augmentation in ACE activity. In addition, this effect could be blocked by the B2 receptor antagonist icatibant. The hypothesis was also tested in endothelial cells, a more physiological system, where both proteins are naturally expressed. Endothelial cells from genetically ablated kinin B2 receptor mice showed a decreased ACE activity when compared with wild-type mice cells. In summary, this is the first report showing that the ACE/kinin B2 receptor interaction modulates ACE activity. Taking into account the interplay among ACE, ACE inhibitors, and kinin receptors, we believe that these results will shed new light into the arena of the controversial search for the mechanism controlling these interactions.

Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion.

Analogues containing the paramagnetic amino acid TOAC as substrates for angiotensin I-converting enzyme

The angiotensin I‐converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C‐terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC1‐Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC3‐Ang I were cleaved by ACE. Quenching of Tyr4 fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.

Theoretical Basis, Laboratory Evidence, and Clinical Research of Chemical Surgery of the Cornea: Cross-Linking

Corneal cross-linking (CXL) is increasingly performed in ophthalmology with high success rates for progressive keratoconus and other types of ectasia. Despite being an established procedure, some molecular and clinical aspects still require additional studies. This review presents a critical analysis of some established topics and others that are still controversial. In addition, this review examines new technologies and techniques (transepithelial and ultrafast CXL), uses of corneal CXL including natural products and biomolecules as CXL promoters, and evidence for in vitro and in vivo indirect effectiveness.

Enzima conversora de angiotensina no líquido pericárdico: estudo comparativo com a atividade sérica

BACKGROUND The characterization of an angiotensin-converting enzyme (ACE) in human pericardial fluid is relevant, considering its role in the angiotensin II release and thus, the role of the pericardium in cardiovascular homeostasis. OBJECTIVE To isolate and characterize an ACE from human pericardial fluid and to compare the angiotensin I converting activities of the pericardial fluid with that of the serum in patients submitted to cardiovascular surgery. METHODS The enzyme from human pericardial fluid was purified through chromatographic steps and characterized by polyacrylamide gel electrophoresis (SDS-PAGE), hydrolysis of angiotensin I, bradykinin, Hip-His-Leu and synthetic substrates with internal fluorescence suppression. Lisinopril was used as inhibitor. The ACE activity was measured in blood and pericardial fluid samples of 23 patients submitted to cardiovascular surgery. RESULTS The purified ACE (MM = 140 kDa), releases angiotensin II, hydrolyses bradykinin and the Hip-His-Leu substrate. The kinetic parameters k cat,(s-1) and k cat/Km (microM-1. s-1) were, respectively: Hip-His-Leu (1.14 and 7 x 10 -4) ; Abz-YRK(Dnp)P-OH (2.60 and 0.77), Abz-LFK(Dnp)-OH (2.77 and 0.36) and Abz-SDK(Dnp)P-OH (1.92 and 0.19). The angiotensin I converting activities (mean +/- SD) in the pericardial fluid and in blood, were, respectively: 3.16 +/- 0.90 mU x mg -1x min-1 and 0.33 +/- 0.11 mU x mg -1x min-1. The difference was significant between the two fluids. CONCLUSION An ACE that bears great similarity with the somatic enzyme was isolated from human pericardial fluid. The angiotensin I converting activity is higher in the pericardial fluid when compared to the serum activity. These data are important evidence of the role of the pericardial fluid in the metabolism of active peptides.

Conditions for obtaining polyvinyl alcohol/trisodium trimetaphosphate hydrogels as vitreous humor substitute

Hydrogels are polymeric materials with numerous medical and biological applications because of their physicochemical properties. In this context, the conditions were defined for obtaining a hydrogel with characteristics similar to the vitreous humor using polyvinyl alcohol (PVA) and trisodium trimetaphosphate (STMP). The concentration of PVA (X1 ), PVA/STMP ratio (X2 ), and initial pH (X3 ) were modified, and their effect was analyzed in terms of the refractive index (Y1 ), density (Y2 ), dynamic viscosity (Y3 ), and final pH (Y4 ). The results demonstrated that X1 interferes with Y1 , Y2 , and Y3 , and X2 interferes with Y2 and Y3 . The best condition for obtaining a hydrogel with characteristics similar to the vitreous humor was 4.2586% PVA (wt/wt), STMP/PVA ratio of 1:6.8213 (wt/wt), and initial pH of 9.424. DSC, ATR-FTIR, swelling degree, and AFM analysis confirmed the PVA reticulation with STMP. Furthermore, STMP increased the glass transition temperature and decreased the water uptake of ∼50% of the hydrogels, which can be explained by the crosslinking of PVA chains. Infrared spectroscopy revealed a decrease of hydroxyl bonds and confirmed the reticulation between PVA and STMP.

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

Characterization of Rabbit Corneas Subjected to Stromal Stiffening by the Açaí Extract ( Euterpe oleracea )

ABSTRACT Purpose: In this study, we characterized rabbit corneas subjected to corneal cross-linking (CXL) with açaí extract compared with a riboflavin photo-stimulated procedure. Materials and Methods: The corneas of the slaughterhouse rabbits were divided into three groups: control, consisting of untreated corneal samples; riboflavin/UVA, where corneas were treated with 0.1% riboflavin photo-stimulated at 365 nm as the standard protocol; and açaí, where the samples were subjected to 4% açaí extract for 0.5–2 h. After the CXL procedure, corneas of the three groups were characterized by analyzing their elastic modulus and thermal denaturation profile. Results: The elastic modulus at 3% strain showed an approximately threefold increase in the riboflavin/UVA group and 10.5 times in the corneas treated with 4% açaí extract for 2 h, compared with the control group (p < 0.01). The denaturation temperature values of the two groups of crosslinked corneas increased significantly (p < 0.05) and were more pronounced in the açaí group. Conclusions: The açaí extract was effective in promoting CXL in rabbit corneas as characterized by the different techniques.

Characterization of PVA/glutaraldehyde hydrogels obtained using Central Composite Rotatable Design (CCRD)

Hydrogels are made from natural or synthetic polymers and, currently, they have many biomedical applications. In this work, the conditions for obtaining a hydrogel with similar physicochemical characteristics to the vitreous humor were defined using polyvinyl alcohol (PVA) and glutaraldehyde (GLUT) as cross-linker. The concentration of PVA and GLUT were modified, and their effect was analyzed in terms of the refractive index, density, and dynamic viscosity. The hydrogel which was obtained using 3.98% (w/V) of PVA, 3.13 mL (1.57 g) of GLUT in 100 mL, and the initial pH of 7.2 showed similar characteristics to the vitreous humor (density = 1.0174 ± 0.0050 g mL-1 , dynamic viscosity = 3.7425 ± 0.1800 mPa s and refractive index = 1.3410 ± 0.0010). The hydrogels were further investigated by rheological measurements, infrared spectroscopy, differential scanning calorimetry, X-ray diffraction and determination of swelling degree. The reticulation with GLUT promoted an increase in viscosity and glass transition temperature. On the other hand, it stimulated a decrease in the swelling degree, crystallinity, melting temperature, and intensity of the band related to the -OH bond, compared with the PVA without reticulation. The reticulated hydrogel displayed Newtonian behavior and a higher apparent viscosity than the PVA.

Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies

This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac0-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac3-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9μM-1min-1 for BK and Toac3-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.