Secreted Extracellular Cyclophilin A Is a Novel Mediator of Ventilator-induced Lung Injury.

Rationale: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator-induced lung injury. eCypA (extracellular CypA [cyclophilin A]) is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to coronavirus disease (COVID-19). Objectives: To explore the involvement of eCypA in the pathophysiology of ventilator-induced lung injury. Methods: Mice were ventilated with a low or high Vt for up to 3 hours, with or without blockade of eCypA signaling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretching to explore the cellular source of eCypA, and CypA concentrations were measured in BAL fluid from patients with acute respiratory distress syndrome to evaluate the clinical relevance. Measurements and Main Results: High-Vt ventilation in mice provoked a rapid increase in soluble CypA concentration in the alveolar space but not in plasma. In vivo ventilation and in vitro stretching experiments indicated the alveolar epithelium as the likely major source. In vivo blockade of eCypA signaling substantially attenuated physiological dysfunction, macrophage activation, and MMPs (matrix metalloproteinases). Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated concentrations of eCypA within BAL fluid. Conclusions: CypA is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. eCypA represents an exciting novel target for pharmacological intervention.

Cyclophilin A inhibition as potential treatment of human aortic valve calcification.

Aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification process of AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that (i) CyPA is up-regulated in stenotic valves of AS patients, (ii) pro-calcifying medium promotes CyPA secretion by VICs, (iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and (iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.

Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival in a Mouse Model of Amyotrophic Lateral Sclerosis.

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1G93A mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1G93A mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENT We provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1G93A mouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.

Screening for Selective Protein Inhibitors by Using the IANUS Peptide Array.

Finding new road blacks: A peptidic inhibitor of calcineurin (CaN)-mediated nuclear factor of activated T cells (NFAT) dephosphorylation, which is developed through a template-assisted IANUS (Induced orgANisation of strUcture by matrix-assisted togethernesS) peptide array, is cell permeable and able to block the translocation of green fluorescent protein-NFAT fusion protein (GFP-NFAT) into the nucleus after stimulation.

Synthesis and biochemical evaluation of two novel N-hydroxyalkylated cyclosporin A analogs.

The cyclic undecapeptide cyclosporin A (CsA) is a widely used immunosuppressive agent. Its immunosuppressive properties arise from strong binding to cyclophilins (Cyp) followed by inhibition of the protein calcineurin (CaN) by the binary CsA/Cyp complex and subsequent negative regulation of T-cell activation. In the present study we show a novel way to modify the CsA ring by selective N-hydroxyalkylation of the residues Val5 and d-Ala8. Moreover, the influence of these structural CsA modifications on the ability of the CsA analogs to bind Cyp, to inhibit CaN and to penetrate membranes of living cells was investigated. Our results show that the Val5 N-substitution significantly improved compound cell-permeability and markedly diminished CaN inhibition by the binary CsA analog/CypA complex but to a lesser extent Cyp inhibition. In contrast, the N-alkylation of d-Ala8 gave a product with significantly reduced affinity for Cyp but its immunosuppressive effects remained similar to CsA. Possible explanations of the observed experimental data are provided by computational studies.

Inhibition of Aß(1-40) fibril formation by cyclophilins.

Cyclophilins interact directly with the Alzheimer's disease peptide Aß (amyloid ß-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aß binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aß(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aß(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aß(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aß(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aß fibril formation by cyclophilins.

Identification of low abundance cyclophilins in human plasma.

Cylophilins (Cyps) belong to the ubiquitously distributed enzyme class of peptidyl prolyl cis/trans isomerases (EC5.2.1.8), which are foldases capable of accelerating slow steps in the refolding of denatured proteins. At least 20 different Cyp isoenzymes are broadly distributed among all organs and cellular compartments in humans. Extracellularly localized Cyps came into the scientific focus recently because of their involvement in the control of inflammatory diseases, as well as viral and bacterial infections. However, detailed insights into Cyp functions are often hampered by the lack of sensitive detection methods. We present an improved method for affinity purification and detection of Cyp in biotic samples in this manuscript. The procedure takes advantage of two novel cyclosporine A derivatives. Derivative 1 was used to capture Cyps from the sample while derivative 2 was applied for selective release from the affinity matrix. Using this approach, eight different Cyp (CypA, CypB, CypC, Cyp40 (PPID), CypE, CypD (PPIF), CypH, and CypL1) were unambiguously detected in healthy human blood plasma. Moreover, extracellular CypA was found to be partially modified by Nε acetylation on residues Lys44, Lys133, Lys155, as well as Nα  acetylation at the N-terminal Val residue. Nα  acetylation of Ser2 residue was also found for Cyp40.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

Targeting Extracellular Cyclophilins Ameliorates Disease Progression in Experimental Biliary Atresia.

Biliary atresia (BA) is a devastating liver disease of unknown etiology affecting children generally within the first 3 months of life. The disease is manifested by inflammation and subsequent obstruction of the extrahepatic bile ducts, fibrosis and liver failure. The mechanisms responsible for disease pathogenesis are not fully understood, but a number of factors controlled by the SMAD signaling pathway have been implicated. In this study, we investigated the role of a known proinflammatory factor, extracellular cyclophilin A (CypA), in the pathogenesis of biliary atresia using the rhesus rotavirus (RRV) murine model. We used a unique cyclosporine A derivative, MM284, which does not enter cells and therefore inactivates exclusively extracellular cyclophilins, as a potential treatment. We demonstrated that levels of CypA in plasma of RRV-infected mice were increased significantly, and that treatment of mice with MM284 prior to or one day after disease initiation by RRV infection significantly improved the status of mice with experimental BA: weight gain was restored, bilirubinuria was abrogated, liver infiltration by inflammatory cells was reduced and activation of the SMAD pathway and SMAD-controlled fibrosis mediators and tissue inhibitor of metalloproteinases (TIMP)-4 and matrix metalloproteinase (MMP)-7 was alleviated. Furthermore, treatment of human hepatic stellate cells with recombinant cyclophilin recapitulated SMAD2/3 activation, which was also suppressed by MM284 treatment. Our data provide the first evidence that extracellular cyclophilins activate the SMAD pathway and promote inflammation in experimental BA, and suggest that MM284 may be a promising therapeutic agent for treating BA and possibly other intrahepatic chronic disorders.

Inhibition of extracellular cyclophilins with cyclosporine analog and development of atherosclerosis in apolipoprotein E-deficient mice.

Cyclophilins exert both intracellular and extracellular activities related to immune responses and inflammation, which have been implicated in pathogenesis of atherosclerosis. Pan-inhibition of cyclophilins has both pro- and antiatherosclerotic properties, but specific contributions of extracellular and intracellular cyclophilins to these effects have not been characterized. Here, using selective inhibitor of extracellular cyclophilins, we investigated the role of these molecules in atherosclerosis. Apolipoprotein E-null mice fed a high-fat diet received intraperitoneal injections every second day of either vehicle or two analogs of cyclosporine A (CsA): [Melle](4)-CsA (NIM811), a nonimmunosupressive cell-permeable inhibitor of both intracellular and extracellular cyclophilins; and [(4R)-4-[(6-carboxy-1H-benzo[d]imidazol-2-yl)-methyl]-4-methyl-l-threonine](1)-CsA (MM284), cell-impermeable analog only inhibiting extracellular cyclophilins. Development of atherosclerosis and composition of plaques in aorta and innominate artery were studied. Both analogs increased abundance and cross-sectional size of the atherosclerotic plaques in aorta but did not affect development of atherosclerosis in innominate artery. Neither compound affected abundance of macrophages and amount of vascular cell adhesion molecule-1 or nitrotyrosine in the plaques of both arteries. Both compounds reduced the amount of collagen in innominate artery without affecting abundance of collagen in aortic sinus. MM284, but not NIM811, significantly reduced plasma concentration of tumor necrosis factor-α (TNFα); neither compound affected plasma concentrations of interleukin (IL)-6, IL-10 or monocyte chemoattractant protein-1. Ratio between different populations of immune cells in blood or isolated from lymph nodes and spleen as well as plasma lipoprotein profile were unaffected by both compounds. In conclusion, selective inhibition of extracellular cyclophilins reduced TNFα levels in plasma but increased atherosclerosis.