Mechanisms of COVID-19-induced kidney injury and current pharmacotherapies.

The COVID-19 pandemic created a worldwide debilitating health crisis with the entire humanity suffering from the deleterious effects associated with the high infectivity and mortality rates. While significant evidence is currently available online and targets various aspects of the disease, both inflammatory and noninflammatory kidney manifestations secondary to COVID-19 infection are still largely underrepresented. In this review, we summarized current knowledge about COVID-19-related kidney manifestations, their pathologic mechanisms as well as various pharmacotherapies used to treat patients with COVID-19. We also shed light on the effect of these medications on kidney functions that can further enhance renal damage secondary to the illness.

Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics.

Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which labeling chemistry is designed. In practice, only two functional groups are present in every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative alkylation for peptide mass spectrometry and single-molecule protein sequencing that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino-acid residues for decarboxylative labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid-labeling technology in tandem to establish a new platform for fluorosequencing.

Rapid and reversible hydrazone bioconjugation in cells without the use of extraneous catalysts.

The amenability of hydrazone linkages to disassemble via either hydrolysis in mildly acidic aqueous solutions or transimination upon treatment with amine nucleophiles renders them extremely attractive for applications in chemical biology, drug delivery and materials science. Unfortunately, however, the use of hydrazones is hampered by the extremely slow intrinsic rates of their formation from their hydrazine and carbonyl precursors. Consequently, hydrazone formation is typically performed in the presence of a large excess of cytotoxic aniline-based nucleophilic catalysts, rendering hydrazones unsuitable for biological applications that entail their formation in cells. Herein, we report a hydrazine scaffold-o-amino benzyl hydrazine-that rapidly forms hydrazones via intramolecular nucleophilic catalysis, thereby obviating the use of extraneous catalysts. We demonstrate the use of this scaffold for rapid and reversible peptide and protein hydrazone bioconjugation and also for reversible fluorescent labeling of sialylated glycoproteins and choline lipids in mammalian cells.

Renin angiotensin system and its role in biomarkers and treatment in gliomas.

Gliomas are the most common primary intrinsic tumor in the brain and are classified as low- or high-grade according to the World Health Organization (WHO). Patients with high-grade gliomas (HGG) who undergo surgical resection with adjuvant therapy have a mean overall survival of 15 months and 100% recurrence. The renin-angiotensin system (RAS), the primary regulator of cardiovascular circulation, exhibits local action and works as a paracrine system. In the context of this local regulation, the expression of RAS peptides and receptors has been detected in different kinds of tumors, including gliomas. The dysregulation of RAS components plays a significant role in the proliferation, angiogenesis, and invasion of these tumors, and therefore in their outcomes. The study and potential application of RAS peptides and receptors as biomarkers in gliomas could bring advantages against the limitations of current tumoral markers and should be considered in the future. The targeting of RAS components by RAS blockers has shown potential of being protective against cancer and improving immunotherapy. In gliomas, RAS blockers have shown a broad spectrum for beneficial effects and are being considered for use in treatment protocols. This review aims to summarize the background behind how RAS plays a role in gliomagenesis and explore the evidence that could lead to their use as biomarkers and treatment adjuvants.

Cardiovascular effects of small peptides of the renin angiotensin system.

The renin-angiotensin system (RAS) is a unique hormonal cascade which is composed by multiple enzymes and effector peptides. Recently, new peptides presenting biological activity have been discovered, increasing the complexity of the RAS Here, we evaluated the effects of small peptides of the RAS in coronary bed of rats. Firstly, we examined the direct effect of small angiotensinergic peptides [Angiotensin (Ang) -(1-5), Ang-(1-4) Ang-(1-3), and Ang-(1-2)] in coronary vessels. Noteworthy, it was observed that Ang-(1-4), Ang-(1-3), and Ang-(1-2) caused a significant reduction in pressure perfusion. Because Ang-(1-2) was the smallest peptide tested and presented the major effect, we decided to investigate its mechanisms of action. The effect of Ang-(1-2) was partially dependent on the Mas receptor, nitric oxide release and angiotensin-converting enzyme. Importantly, Ang-(1-2) reduced the blood pressure of Wistar rats and SHR Interestingly, SHR presented a more pronounced decrease in blood pressure levels than Wistar rats. Altogether, these data showed that angiotensinergic small peptides hold biological activities in coronary bed of rats.

A Primer to Angiotensin Peptide Isolation, Stability, and Analysis by Nano-Liquid Chromatography with Mass Detection.

The renin-angiotensin system (RAS) is an important element of cardiovascular and renal physiology and targeting the RAS by renin inhibitors, angiotensin (Ang) converting enzyme (ACE) inhibitors and Ang II type 1 receptor antagonists is effective in the treatment of hypertension, heart failure, and atherosclerosis. Quantification of Ang peptides is critical to establish the status of the RAS, but it is challenging due to low Ang peptides concentrations (fmol/mL or fmol/g), abundance of interfering substances, post sampling conversions, and difficulties with the specificity of the assay.In this chapter, we describe a new nano-LC/MS-based methodology for comprehensive, specific, sensitive, and accurate quantification of Ang peptides profile in plasma and tissue. We optimized sample pretreatment method (protein removal (acetonitrile precipitation) followed by solid-phase extraction (C18 silica bonded phase)), chromatographic conditions (reversed-phase nanochromatography with preconcentration), and mass detection (multiple reaction monitoring) of nine peptides: Ang-(1-12), Ang I (1-10), Ang-(1-9), Ang II (1-8), [Ala1]-Ang II, Ang III (2-8), Ang IV (3-8), Ang-(1-7), and [Ala1]-Ang-(1-7). Assessment of plasma and cardiac concentrations of Ang peptides in genetically modified atherosclerotic apolipoprotein E/LDL receptor double knockout (ApoE-/-/LDLR-/-) mice vs. wild types revealed changes in renin-angiotensin system consistent with an overactivation of ACE and impairment of ACE2. The method could be easily adopted for high-throughput analysis and for use in clinical applications such as diagnosis of the RAS abnormalities or monitoring of the RAS inhibition-based therapies.

Detecting Low-Abundance Molecules at Single-Cell Level by Repeated Ion Accumulation in Ion Trap Mass Spectrometer.

Low-abundance metabolites or proteins in single-cell samples are usually undetectable by mass spectrometry (MS) due to the limited amount of substances in single cells. This limitation inspired us to further enhance the sensitivity of commercial mass spectrometers. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is capable of enhancing the sensitivity by selectively and repeatedly accumulating ions in a linear ion trap for up to 25 cycles. The increase in MS sensitivity was positively correlated with the number of repeated cycles. When ions were repeatedly accumulated for 25 cycles, the sensitivity of adenosine triphosphate detection was increased by 22-fold within 1.8 s. Our technique could stably detect low-abundance ions, especially MSn ions, at the single-cell level, such as 5-methylcytosine hydrolyzed from sample equivalent to ∼0.2 MCF7 cell. The strategy presented in this study offers the possibility to aid single-cell analysis by enhancing MS detection sensitivity.

Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing.

Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors have been prospected. In order to endorse this research field we have developed a new tool for ACE ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs was inhibited by lisinopril with a half maximal inhibitory concentration (IC50) of 10nM. At the fishing assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE kinetics determination, inhibition and binder screening.

Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism.

Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Šresolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of ∼10 nm involving ∼8 monosaccharides and ∼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m-1 s-1, its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates.

Screen-printed digital microfluidics combined with surface acoustic wave nebulization for hydrogen-deuterium exchange measurements.

An inexpensive digital microfluidic (DMF) chip was fabricated by screen-printing electrodes on a sheet of polyimide. This device was manually integrated with surface acoustic wave nebulization (SAWN) MS to conduct hydrogen/deuterium exchange (HDX) of peptides. The HDX experiment was performed by DMF mixing of one aqueous droplet of angiotensin II with a second containing various concentrations of D2O. Subsequently, the degree of HDX was measured immediately by SAWN-MS. As expected for a small peptide, the isotopically resolved mass spectrum for angiotensin revealed that maximum deuterium exchange was achieved using 50% D2O. Additionally, using SAWN-MS alone, the global HDX kinetics of ubiquitin were found to be similar to published NMR data and back exchange rates for the uncooled apparatus using high inlet capillary temperatures was less than 6%.

Novel renin inhibitors containing derivatives of N-alkylleucyl-ß-hydroxy-γ-amino acids.

In search for new drugs lowering arterial blood pressure, which could be applied in anti-hypertensive therapy, research concerning agents blocking of renin-angiotensin-aldosteron system has been conducted. Despite many years of research conducted at many research centers around the world, aliskiren is the only one renin inhibitor, which is used up to now. Four novel potential renin inhibitors, having structure based on the peptide fragment 8-13 of human angiotensinogen, a natural substrate for renin, were designed and synthesized. All these inhibitors contain unnatural moieties that are derivatives of N-methylleucyl-ß-hydroxy-γ-amino acids at the P2-P1' position: 4-[N-(N-methylleucyl)-amino]-3-hydroxy-7-(3-nitroguanidino)-heptanoic acid (AHGHA), 4-[N-(N-methylleucyl)-amino]-3-hydroxy-5-phenyl-pentanoic acid (AHPPA) or 4-[N-(N-methylleucyl)-amino]-8-benzyloxycarbonylamino-3-hydroxyoctanoic acid (AAHOA). The previously listed synthetic ß-hydroxy-γ-amino acids constitute pseudodipeptidic units that correspond to the P1-P1' position of the inhibitor molecule. An unnatural amino acid, 4-methoxyphenylalanin (Phe(4-OMe)), was introduced at the P3 position of the obtained compounds. Three of these compounds contain isoamylamide of 6-aminohexanoic acid (ε-Ahx-Iaa) at the P2'-P3' position. The proposed modifications of the selected human angiotensinogen fragment are intended to increase bioactivity, bioavailability, and stability of the inhibitor molecule in body fluids and tissues. The inhibitor Boc-Phe(4-OMe)-MeLeu-AHGHA-OEt was obtained in the form of an ethyl ester. The hydrophobicity coefficient, expressed as log P varied between 3.95 and 8.17. In vitro renin inhibitory activity of all obtained compounds was contained within the range 10(-6)-10(-9) M. The compound Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa proved to be the most active (IC50 = 1.05 × 10(-9) M). The compounds Boc-Phe(4-OMe)-MeLeu-AHGHA-Ahx-Iaa and Boc-Phe(4-OMe)-MeLeu-AHPPA-Ahx-Iaa are resistant to chymotrypsin.

Formaldehyde scavengers function as novel antigen retrieval agents.

Antigen retrieval agents improve the detection of formaldehyde-fixed proteins, but how they work is not well understood. We demonstrate that formaldehyde scavenging represents a key characteristic associated with effective antigen retrieval; under controlled temperature and pH conditions, scavenging improves the typical antigen retrieval process through reversal of formaldehyde-protein adduct formation. This approach provides a rational framework for the identification and development of more effective antigen retrieval agents.

Tyrosine-Specific Chemical Modification with in Situ Hemin-Activated Luminol Derivatives.

Tyrosine-specific chemical modification was achieved using in situ hemin-activated luminol derivatives. Tyrosine residues in peptide and protein were modified effectively with N-methylated luminol derivatives under oxidative conditions in the presence of hemin and H2O2. Both single and double modifications of the tyrosine residue occurred in the reaction of angiotensin II with N-methylated luminol derivative 9. Tyrosine-specific chemical modification of the model protein bovine serum albumin (BSA) revealed that the surface-exposed tyrosine residues were selectively modified with 9. We succeeded in the functionalization of several proteins using azide-conjugated compound 18 using alkyne-conjugated probes by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or dibenzocyclooctyne (DBCO)-mediated copper-free click chemistry. This tyrosine-specific modification was orthogonal to conventional lysine modification by N-hydroxysuccinimide (NHS) ester, and dual functionalization by fluorescence modification of tyrosine residues and PEG modification of lysine residues was achieved without affecting the modification efficiency.

Probiotics (VSL#3) prevent endothelial dysfunction in rats with portal hypertension: role of the angiotensin system.

AIMS: Portal hypertension characterized by generalized vasodilatation with endothelial dysfunction affecting nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) has been suggested to involve bacterial translocation and/or the angiotensin system. The possibility that ingestion of probiotics prevents endothelial dysfunction in rats following common bile duct ligation (CBDL) was evaluated. METHODS: Rats received either control drinking water or the probiotic VSL#3 solution (50 billion bacteria.kg body wt⁻¹.day⁻¹) for 7 weeks. After 3 weeks, rats underwent surgery with either resection of the common bile duct or sham surgery. The reactivity of mesenteric artery rings was assessed in organ chambers, expression of proteins by immunofluorescence and Western blot analysis, oxidative stress using dihydroethidium, and plasma pro-inflammatory cytokine levels by flow cytometry. RESULTS: Both NO- and EDH-mediated relaxations to acetylcholine were reduced in the CBDL group compared to the sham group, and associated with a reduced expression of Cx37, Cx40, Cx43, IKCa and SKCa and an increased expression of endothelial NO synthase (eNOS). In aortic sections, increased expression of NADPH oxidase subunits, angiotensin converting enzyme, AT1 receptors and angiotensin II, and formation of ROS and peroxynitrite were observed. VSL#3 prevented the deleterious effect of CBDL on EDH-mediated relaxations, vascular expression of connexins, IKCa, SKCa and eNOS, oxidative stress, and the angiotensin system. VSL#3 prevented the CBDL-induced increased plasma TNF-α, IL-1α and MCP-1 levels. CONCLUSIONS: These findings indicate that VSL#3 ingestion prevents endothelial dysfunction in the mesenteric artery of CBDL rats, and this effect is associated with an improved vascular oxidative stress most likely by reducing bacterial translocation and the local angiotensin system.

Characterization of the enhanced peroxidatic activity of amyloid ß peptide-hemin complexes towards neurotransmitters.

Binding of heme to the amyloid peptides Aß40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer's disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the binding of hemin to Aß peptides might play an even more crucial role in the early stages of Alzheimer's disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide Aß1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone.

Laser-induced dissociation of singly protonated peptides at 193 and 266 nm within a hybrid linear ion trap mass spectrometer.

RATIONALE: We implemented, for the first time, laser-induced dissociation (LID) within a modified hybrid linear ion trap mass spectrometer, QTrap, while preserving the original scanning capabilities and routine performance of the instrument. METHODS: Precursor ions of interest were mass-selected in the first quadrupole (Q1), trapped in the radiofrequency-only quadrupole (q2), photodissociated under irradiation with a 193- or 266-nm laser beam in the third quadrupole (q3), and mass-analyzed using the linear ion trap. RESULTS: LID of singly charged protonated peptides revealed, in addition to conventional amide-bond cleavages, preferential fragmentation at Cα -C/N-Cα bonds of the backbone as well as at the Cα -Cß /Cß -Cγ bonds of the side-chains. The LID spectra of [M+H](+) featured product ions that were very similar to the observed radical-induced fragmentations in the CID spectra of analogous odd-electron radical cations generated through dissociative electron-transfer in metal-ligand-peptide complexes or through laser photolysis of iodopeptides. CONCLUSIONS: LID of [M+H](+) ions results in fragmentation channels that are comparable with those observed upon the CID of M(•+) ions, with a range of fascinating radical-induced fragmentations.

Investigation of ethanol-peptide and water-peptide interactions through intermolecular nuclear overhauser effects and molecular dynamics simulations.

Molecular dynamics simulations have been used to explore solvent-solute intermolecular nuclear Overhauser effects (NOEs) on NMR (nuclear magnetic resonance) signals of [val5]angiotensin dissolved in 35% ethanol-water (v/v). Consideration of chemical shift, coupling constant and intramolecular NOE data suggest that conformations of the peptide are adequately sampled by simulations of up to 0.6 µs duration. Calculated cross relaxation terms at 0 and 25 °C are compared to experimental values and to terms predicted using a particulate model of the solvent. Many calculated solvent NOEs are in agreement with experimental results; disagreements are particularly striking for hydrogens of the Phe8 residue of the peptide. Calculations show that individual molecules of either solvent component can spend many ns in association with the peptide but dipolar interactions within such a complex account for only a few percent of an observed cross relaxation rate. Most parts of the peptide interact selectively with ethanol. Diffusion of both solvent components is slowed when they are close to the peptide. Solvent-solute cross relaxation terms for acetic acid in the same solvent obtained from simulations agree with experiment. Preferential interactions of solvent molecules with acetic acid are largely absent, as are effects of this solute on solvent diffusion rates.

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin.

A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10 µM) and PD123319 (10 µM) plus 100 µM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50s ~1-20 µM. This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100 µM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-Hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [45] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding.

The X-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.

Human aminopeptidase N (hAPN/hCD13) is a dimeric membrane protein and a member of the M1 family of zinc metallopeptidases. Within the rennin-angiotensin system, its enzymatic activity is responsible for processing peptide hormones angiotensin III and IV. In addition, hAPN is also involved in cell adhesion, endocytosis, and signal transduction and it is an important target for cancer therapy. Reported here are the high resolution x-ray crystal structures of the dimeric ectodomain of hAPN and its complexes with angiotensin IV and the peptidomimetic inhibitors, amastatin and bestatin. Each monomer of the dimer is found in what has been termed the closed form in other M1 enzymes and each monomer is characterized by an internal cavity surrounding the catalytic site as well as a unique substrate/inhibitor-dependent loop ordering, which in the case of the bestatin complex suggests a new route to inhibitor design. The hAPN structure provides the first example of a dimeric M1 family member and the observed structural features, in conjunction with a model for the open form, provide novel insights into the mechanism of peptide processing and signal transduction.

The effects of angiotensin peptides and angiotensin receptor antagonists on the cell growth and angiogenic activity of GH3 lactosomatotroph cells in vitro.

The local renin-angiotensin system (RAS) is present in the pituitary gland, and inhibitory effects of angiotensins on the lactosomatotroph (GH3) cell growth have been revealed. The aim of this study was to examine the influence of various angiotensin peptides and angiotensin AT1, AT2, and AT4 receptors antagonists on the cell proliferation, viability, and VEGF secretion in pituitary lactosomatotroph GH3 cell culture in order to identify receptors involved in antiproliferative effects of angiotensins on GH3 tumor cells. Cell viability and proliferation using Mosmann method and BrdU incorporation during DNA synthesis, and VEGF secretion using ELISA assay were estimated. The inhibitory effects of ang II, ang IV, and ang 5-8 on the cell viability and BrdU incorporation in GH3 culture were not abolished by AT1, AT2, and AT4 receptors antagonists. Ang II, as well as ang III and ang IV at lower concentrations stimulated the secretion of VEGF in GH3 cell culture. The secretion of VEGF was inhibited by ang III and ang IV at higher concentrations. AT1 and AT2 receptors antagonists prevented the proangiogenic effects of ang II. Ang II, ang IV, and ang 5-8 decrease the cell number and proliferation in GH3 cell culture independently of the AT1, AT2, and AT4 receptors. These peptides affect also secretion of VEGF in culture examined. Both the AT1 and AT2 receptors appear to mediate the proangiogenic effects of ang II.