Conformational Changes of Blood ACE in Chronic Uremia

Petrov, M. N.; Shilo, V. Yu.; Tarasov, Alexandr V.; Schwartz, David E.; Garcia, Joe G.N.; Кост, О. А.; Danilov, Sergei M.

doi:10.1371/journal.pone.0049290

Cited by 16 publications

(34 citation statements)

References 78 publications

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“…To our knowledge, this is the first report demonstrating uremic elevation of mACE-expression in relation to pro-atherogenic behaviour of primary human monocytes or THP-1 cells. Such a potential of ACE was previously reported in animal and cell culture models but was not directly correlated with influences of uremia on human monocytes in vitro [26], [27], [28], [29]. With regards to other cell culture systems, it has been demonstrated that uremic toxins may affect oxidative burst activity of the leukocytes and increase their pro-inflammatory effects.…”

Section: Discussionmentioning

confidence: 80%

Uremic Conditions Drive Human Monocytes to Pro-Atherogenic Differentiation via an Angiotensin-Dependent Mechanism

et al. 2014

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AimsElevated expression levels of monocytic-ACE have been found in haemodialysis patients. They are not only epidemiologically linked with increased mortality and cardiovascular disease, but may also directly participate in the initial steps of atherosclerosis. To further address this question we tested the role of monocytic-ACE in promotion of atherosclerotic events in vitro under conditions mimicking those of chronic renal failure.Methods and ResultsTreatment of human primary monocytes or THP-1 cells with uremic serum as well as PMA-induced differentiation led to significantly up-regulated expression of ACE, further increased by additional treatment with LPS. Functionally, these monocytes revealed significantly increased adhesion and transmigration through endothelial monolayers. Overexpression of ACE in transfected monocytes or THP-1 cells led to development of more differentiated, macrophage-like phenotype with up-regulated expression of Arg1, MCSF, MCP-1 and CCR2. Expression of pro-inflammatory cytokines TNFa and IL-6 were also noticeably up-regulated. ACE overexpression resulted in significantly increased adhesion and transmigration properties. Transcriptional screening of ACE-overexpressing monocytes revealed noticeably increased expression of Angiotensin II receptors and adhesion- as well as atherosclerosis-related ICAM-1 and VCAM1. Inhibition of monocyte ACE or AngII-receptor signalling led to decreased adhesion potential of ACE-overexpressing cells.ConclusionsTaken together, these data demonstrate that uremia induced expression of monocytic-ACE mediates the development of highly pro-atherogenic cells via an AngII-dependent mechanism.

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Section: Discussionmentioning

confidence: 80%

Uremic Conditions Drive Human Monocytes to Pro-Atherogenic Differentiation via an Angiotensin-Dependent Mechanism

et al. 2014

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“…In order to save valuable mAbs, we used not more than 3 μg/ml of pure mAbs (or 1/20 dilution of hybridoma cell culture medium) which was found to be sufficient for coating (Fig 1C and 1D). Usually the amount of ACE immunoreactive protein was estimated using the strongest mAb to ACE, clone 9B9 [16, 20], while a pattern of ACE activity precipitation by a panel of mAbs to different epitopes on ACE—conformational fingerprint of ACE—was used for the estimation of local conformational differences in ACE surface topography due to disease [5, 7, 21] or due to tissue origin of ACE [5, 9, 10].…”

Section: Resultsmentioning

confidence: 99%

“…Relative precipitation of ACE by strong mAbs (9B9, 2B11, 3A5, 2H9) dramatically differed compared to precipitation by weak mAbs (i1A8, 3G8, 1E10, and 3F11), likely reflecting difference in their affinity to ACE (Fig 7A). We should mention that some mAbs demonstrated anti-catalytic effect on ACE in solution at μg/ml concentrations [6, 15], whereas in the enzyme immunocapture assay (at an order lower concentrations the anti-catalytic effect did not exceed 20% (S1 Fig in [7]) in comparison with 10-fold differences in fluorescence signals demonstrated by ACE precipitated by different mAbs, weak and strong (Fig 7A). Therefore, we can consider anti-catalytic effect of some mAbs in this format as negligible.…”

Section: Resultsmentioning

confidence: 99%

Conformational fingerprint of blood and tissue ACEs: Personalized approach

et al. 2018

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BackgroundThe pattern of binding of monoclonal antibodies (mAbs) to 18 epitopes on human angiotensin I-converting enzyme (ACE)–“conformational fingerprint of ACE”–is a sensitive marker of subtle conformational changes of ACE due to mutations, different glycosylation in various cells, the presence of ACE inhibitors and specific effectors, etc.Methodology/Principal findingsWe described in detail the methodology of the conformational fingerprinting of human blood and tissue ACEs that allows detecting differences in surface topography of ACE from different tissues, as well detecting inter-individual differences. Besides, we compared the sensitivity of the detection of ACE inhibitors in the patient’s plasma using conformational fingerprinting of ACE (with only 2 mAbs to ACE, 1G12 and 9B9) and already accepted kinetic assay and demonstrated that the mAbs-based assay is an order of magnitude more sensitive. This approach is also very effective in detection of known (like bilirubin and lysozyme) and still unknown ACE effectors/inhibitors which nature and set could vary in different tissues or different patients.Conclusions/SignificancePhenotyping of ACE (and conformational fingerprinting of ACE as a part of this novel approach for characterization of ACE) in individuals really became informative and clinically relevant. Appreciation (and counting on) of inter-individual differences in ACE conformation and accompanying effectors make the application of this approach for future personalized medicine with ACE inhibitors more accurate. This (or similar) methodology can be applied to any enzyme/protein for which there is a number of mAbs to its different epitopes.

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“…First of all, we demonstrated that the binding of some mAbs was extremely sensitive to local changes in ACE conformation-due to local denaturation or inactivation, binding of ACE inhibitors or antibodies to ACE, or due to several diseases [34,39,40,[43][44][45].…”

Section: Generation Of Monoclonal Antibodies To Human Acementioning

confidence: 97%

“…It turned out that the conformationally altered ACE is characterized by an increased (up to 4 times) activity with respect to the natural substrate angiotensin I, less efficiently inhibited by a specific ACE inhibitor enalaprilat (tripeptide analogue widely used as an antihypertensive agent) and practically was not inhibited by another ACE inhibitor-nonapeptide teprotide. We also analyzed a large sample of blood plasma from healthy donors [48] and patients without uremia [62] and found eight cases of the same conformationally altered ACE as with uremia-which are less susceptible to specific ACE inhibitors [45]. Thus, patients with such conformationally altered ACE in their blood should probably be given more intensive therapy with ACE inhibitors, or replace them with antihypertensive agents of another class.…”

Section: Lungmentioning

confidence: 99%

Conformational Fingerprinting Using Monoclonal Antibodies (on the Example of Angiotensin I-Converting Enzyme-ACE)

Danilov

2017

Mol Biol

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⎯During the past 30 years my laboratory has generated 40+ monoclonal antibodies (mAbs) directed to structural and conformational epitopes on human ACE as well as ACE from rats, mice and other species. These mAbs were successfully used for detection and quantification of ACE by ELISA, Western blotting, flow cytometry and immunohistochemistry. In all these applications mainly single mAbs were used. We hypothesized that we can obtain a completely new kind of information about ACE structure and function if we use the whole set of mAbs directed to different epitopes on the ACE molecule. When we finished epitope mapping of all mAbs to ACE (and especially, those recognizing conformational epitopes), we realized that we had obtained a new tool to study ACE. First, we demonstrated that binding of some mAbs is very sensitive to local conformational changes on the ACE surface-due to local denaturation, inactivation, ACE inhibitor or mAbs binding or due to diseases. Second, we were able to detect, localize and characterize several human ACE mutations. And, finally, we established a new concept-conformational fingerprinting of ACE using mAbs that in turn allowed us to obtain evidence for tissue specificity of ACE, which has promising scientific and diagnostic perspectives. The initial goal for the generation of mAbs to ACE 30 years ago was obtaining mAbs to organ-specific endothelial cells, which could be used for organ-specific drug delivery. Our systematic work on characterization of mAbs to numerous epitopes on ACE during these years has lead not only to the generation of the most effective mAbs for specific drug/gene delivery into the lung capillaries, but also to the establishment of the concept of conformational fingerprinting of ACE, which in turn gives a theoretical base for the generation of mAbs, specific for ACE from different organs. We believe that this concept could be applicable for any glycoprotein against which there is a set of mAbs to different epitopes.

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Conformational Changes of Blood ACE in Chronic Uremia

Cited by 16 publications

References 78 publications

Uremic Conditions Drive Human Monocytes to Pro-Atherogenic Differentiation via an Angiotensin-Dependent Mechanism

Uremic Conditions Drive Human Monocytes to Pro-Atherogenic Differentiation via an Angiotensin-Dependent Mechanism

Conformational fingerprint of blood and tissue ACEs: Personalized approach

Conformational Fingerprinting Using Monoclonal Antibodies (on the Example of Angiotensin I-Converting Enzyme-ACE)

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