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Introduction: Despite advancements in transplant immunology and vascularized composite allotransplantation (VCA), the longevity of allografts remains hindered by the challenge of allograft rejection. The acute-phase response, an immune-inflammatory reaction to ischemia/reperfusion that occurs directly after allogeneic transplantation, serves as a catalyst for graft rejection. This immune response is orchestrated by acute-phase reactants through intricate crosstalk with the mononuclear phagocyte system., Objective: C-reactive protein (CRP), a well-known marker of inflammation, possesses pro-inflammatory properties and exacerbates ischemia/reperfusion injury. Thus, we investigated how CRP impacts acute allograft rejection., Methods: Prompted by clinical observations in facial VCAs, we employed a complex hindlimb transplantation model in rats to investigate the direct impact of CRP on transplant rejection., Results: Our findings demonstrate that CRP expedites allograft rejection and diminishes allograft survival by selectively activating non-classical monocytes. Therapeutic stabilization of CRP abrogates this activating effect on monocytes, thereby attenuating acute allograft rejection. Intravital imagining of graft-infiltrating, recipient-derived monocytes during the early phase of acute rejection corroborated their differential regulation by CRP and their pivotal role in driving the initial stages of graft rejection., Conclusion: The differential activation of recipient-derived monocytes by CRP exacerbates the innate immune response and accelerates clinical allograft rejection. Thus, therapeutic targeting of CRP represents a novel and promising strategy for preventing acute allograft rejection and potentially mitigating chronic allograft rejection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

C-reactive protein (CRP) is an early-stage acute phase protein and highly upregulated in response to inflammatory reactions. We recently identified a novel mechanism that leads to a conformational change from the native, functionally relatively inert, pentameric CRP (pCRP) structure to a pentameric CRP intermediate (pCRP*) and ultimately to the monomeric CRP (mCRP) form, both exhibiting highly pro-inflammatory effects. This transition in the inflammatory profile of CRP is mediated by binding of pCRP to activated/damaged cell membranes via exposed phosphocholine lipid head groups. We designed a tool compound as a low molecular weight CRP inhibitor using the structure of phosphocholine as a template. X-ray crystallography revealed specific binding to the phosphocholine binding pockets of pCRP. We provide in vitro and in vivo proof-of-concept data demonstrating that the low molecular weight tool compound inhibits CRP-driven exacerbation of local inflammatory responses, while potentially preserving pathogen-defense functions of CRP. The inhibition of the conformational change generating pro-inflammatory CRP isoforms via phosphocholine-mimicking compounds represents a promising, potentially broadly applicable anti-inflammatory therapy., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

CRP is an important mediator of the inflammatory response. Pro-inflammatory CRP effects are mediated by pCRP* and mCRP, dissociation products of the native pCRP. The concentration of pCRP during inflammation may rise up to concentrations 1000-fold from baseline. By prevention of the conformational change from pCRP to pCRP*, pro-inflammatory immune responses can be inhibited and local tissue damage reduced. 3-(Dibutylamino)propylphosphonic acid (C10m) is a new substance that can suppress ischemic-reperfusion injury by targeting CRP in the complement cascade. It hampers dissociation of pCRP into its monomers, thus preventing exacerbation of tissue inflammation subsequent to reperfusion injury. In this study, the pharmacokinetics and metabolism of the new drug candidate C10m was investigated. A sensitive and selective method for detection of C10m and its metabolites from plasma and urine was developed with LC-MS and LC-MS/MS coupling. The LLOQ is at 0.1 µg mL -1 and recovery at 87.4% ± 2.8%. Accuracy and precision were within 15% coefficient of variation and nominal concentrations, respectively. Concentration time profile after i.v. bolus injection of C10m was analyzed by LC-MS/MS. Bioavailability has shown to be below 30%. Most likely due to the compounds' very polar chemical properties, no phase-I or phase-II metabolism could be observed. Absence of phase-I metabolism was cross-checked by performing microsomal incubations. Our study revealed that C10m is rapidly eliminated via urine excretion and that half-times appear to be increased with coadministration of the target pCRP., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: C-reactive protein circulates as a pentameric protein (pCRP). pCRP is a well-established diagnostic marker as plasma levels rise in response to tissue injury and inflammation. We recently described pro-inflammatory properties of CRP, which are mediated by conformational changes from pCRP to bioactive isoforms expressing pro-inflammatory neo-epitopes [pCRP* and monomeric C-reactive protein (mCRP)]. Here, we investigate the role of CRP isoforms in renal ischemia/reperfusion injury (IRI)., Methods: Rat kidneys in animals with and without intraperitoneally injected pCRP were subjected to IRI by the time of pCRP exposure and were subsequently analyzed for monocyte infiltration, caspase-3 expression, and tubular damage. Blood urea nitrogen (BUN) was analyzed pre-ischemia and post-reperfusion. CRP effects on leukocyte recruitment were investigated via intravital imaging of rat-striated muscle IRI. Localized conformational CRP changes were analyzed by immunohistochemistry using conformation specific antibodies. 1,6-bis(phosphocholine)-hexane (1,6-bisPC), which stabilizes CRP in its native pentameric form was used to validate CRP effects. Leukocyte activation was assessed by quantification of reactive oxygen species (ROS) induction by CRP isoforms ex vivo and in vitro through electron spin resonance spectroscopy. Signaling pathways were analyzed by disrupting lipid rafts with nystatin and subsequent ROS detection. In order to confirm the translational relevance of our findings, biopsies of microsurgical human free tissue transfers before and after IRI were examined by immunofluorescence for CRP deposition and co-localization of CD68 + leukocytes., Results: The application of pCRP aggravates tissue damage in renal IRI. 1,6-bisPC reverses these effects via inhibition of the conformational change that leads to exposure of pro-inflammatory epitopes in CRP (pCRP* and mCRP). Structurally altered CRP induces leukocyte-endothelial interaction and induces ROS formation in leukocytes, the latter can be abrogated by blocking lipid raft-dependent signaling pathways with Nystatin. Stabilizing pCRP in its native pentameric state abrogates these pro-inflammatory effects. Importantly, these findings are confirmed in human IRI challenged muscle tissue., Conclusion: These results suggest that CRP is a potent modulator of IRI. Stabilizing the native pCRP conformation represents a promising anti-inflammatory therapeutic strategy by attenuation of leukocyte recruitment and ROS formation, the primary pathomechanisms of IRI.

Rationale: The interaction of circulating cells within the vascular wall is a critical event in chronic inflammatory processes, such as atherosclerosis, but the control of the vascular inflammatory state is still largely unclear., Objective: This study was undertaken to characterize the function of the endothelial-enriched microRNA miR-100 during vascular inflammation and atherogenesis., Methods and Results: Based on a transcriptome analysis of endothelial cells after miR-100 overexpression, we identified miR-100 as a potent suppressor of endothelial adhesion molecule expression, resulting in attenuated leukocyte-endothelial interaction in vitro and in vivo as shown by flow cytometry and intravital imaging. Mechanistically, miR-100 directly repressed several components of mammalian target of rapamycin complex 1-signaling, including mammalian target of rapamycin and raptor, which resulted in a stimulation of endothelial autophagy and attenuated nuclear factor κB signaling in vitro and in vivo. In a low-density lipoprotein receptor-deficient atherosclerotic mouse model, pharmacological inhibition of miR-100 resulted in enhanced plaque lesion formation and a higher macrophage content of the plaque, whereas a systemic miR-100 replacement therapy had protective effects and attenuated atherogenesis, resulting in a decrease of plaque area by 45%. Finally, analysis of miR-100 expression in >70 samples obtained during carotid endarterectomy revealed that local miR-100 expression was inversely correlated with inflammatory cell content in patients., Conclusions: In summary, we describe an anti-inflammatory function of miR-100 in the vascular response to injury and inflammation and identify an important novel modulator of mammalian target of rapamycin signaling and autophagy in the vascular system. Our findings of miR-100 as a potential protective anti-athero-miR suggest that the therapeutic replacement of this microRNA could be a potential strategy for the treatment of chronic inflammatory diseases, such as atherosclerosis, in the future., (© 2017 American Heart Association, Inc.)

Background: Adaptive neovascularization after arterial occlusion is an important compensatory mechanism in cardiovascular disease and includes both the remodeling of pre-existing vessels to collateral arteries (arteriogenesis) and angiogenic capillary growth. We now aimed to identify regulatory microRNAs involved in the modulation of neovascularization after femoral artery occlusion in mice., Methods and Results: Using microRNA-transcriptome analysis, we identified miR-155 as a downregulated microRNA during hindlimb ischemia. Correspondingly, inhibition of miR-155 in endothelial cells had a stimulatory effect on proliferation and angiogenic tube formation via derepression of its direct target gene angiotensin II type 1 receptor. Surprisingly, miR-155-deficient mice showed an unexpected phenotype in vivo, with a strong reduction of blood flow recovery after femoral artery ligation (arteriogenesis) dependent on the attenuation of leukocyte-endothelial interaction and a reduction of proarteriogenic cytokine expression. Consistently, miR-155-deficient macrophages exhibit a specific alteration of the proarteriogenic cytokine expression profile, which is partly mediated by the direct miR-155 target gene SOCS-1., Conclusions: Our data demonstrate that miR-155 exerts an antiangiogenic but proarteriogenic function in the regulation of neovascularization via the suppression of divergent cell-specific target genes and that its expression in both endothelial and bone marrow-derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice., (© 2015 American Heart Association, Inc.)