Extracellular and intracellular cyclophilin A, native and post-translationally modified, show diverse and specific pathological roles in diseases

Abstract

Cyclophilin A (CypA) is a ubiquitous and highly conserved protein with peptidyl prolyl isomerase activity. Due to its highly abundant level in the cytoplasm, most studies have focused on the roles of CypA as an intracellular protein. However, emerging evidence suggests an important role for extracellular CypA in the pathogenesis of several diseases through receptor- (CD147 or other) mediated autocrine and paracrine signaling pathways. In this review, we will discuss the shared and unique pathological roles of extracellular and intracellular CypA in human cardiovascular diseases. In addition, the evolving role of post-translational modifications of CypA in the pathogenesis of disease is discussed. Finally, recent studies with drugs specific for extracellular CypA show its importance in disease pathogenesis in several animal models, and make extracellular CypA a new therapeutic target.

Introduction of extracellular and intracellular Cyclophilin A

Cyclophilins are a family of ubiquitous and highly conserved proteins with peptidyl prolyl isomerase (PPIase) activity, which catalyzes the cis-trans isomerization of peptide bonds at proline residues^1–2. Cyclophilin A (CypA) is the most abundantly expressed and first identified cyclophilin among the 18 known human cyclophilins³. It was first identified as the cytosolic binding partner of the immunosuppressive drug cyclosporin A (CsA)⁴. The formation of CypA-CsA complex inhibits the transcription of immune response related genes and prevents proliferation of T cells⁵.

CypA was initially believed to function as an intracellular protein due to its highly abundant level in the nucleus and cytoplasm of many cell types, and its lack of signal export sequence^{3, 6–7}. Indeed, intracellular CypA (iCypA), with its catalytic and chaperone activity of PPIase, has been shown to influence major pathways such as protein folding, trafficking and function. As a consequence of these interactions, it regulates cell function such as T cell subtype differentiation, platelet activation and cytokinesis. CypA has been reported to participate in both de novo protein folding and refolding processes via its PPIase activity^7–10. Several studies have suggested that CypA participated in protein trafficking in cells^11–12. Our lab also reported that iCypA enhanced angiotensin II induced reactive oxygen species (ROS) production in vascular smooth muscle cell (VSMC) by promoting translocation of NADPH oxidase cytosolic subunit p47phox to the caveolae through its interaction with p47phox and cell cytoskeleton¹³. CypA was reported to interact with YY1, a zinc finger transcription factor and alter its transcriptional activity¹⁴. In terms of T cell signaling, Colgan et al. reported that CypA inhibits CD4+ T cell Th2 differentiation through the interaction with Itk via its PPIase active site, causing a favored shift to Th1 profile¹⁵. Wang et al. reported that iCypA promoted platelet activation by inducing calcium influx and facilitating the interaction between αIIbβ3 and cytoskeleton¹⁶. Recently, it was suggested that iCypA was a centrosome protein that undergoes cell cycle-dependent relocation to the midzone and midbody and was required for the completion of cytokinesis¹⁷.

Recent evidence shows an important role for secreted CypA, or extracellular CypA (eCypA), which participates in both autocrine and paracrine signaling pathways^18–19. Reports from Billich et al. showed high levels of CypA in the synovial fluids from patients with rheumatoid arthritis²⁰. Our group was the first to show oxidative stress induced CypA secretion from VSMC. We found that the secretion of CypA from VSMC was a highly regulated process of vesicle transport, docking, and fusion at the plasma membrane²¹, similar to neurotransmitters in the synaptic gap. Substantial evidence showed that many cell types other than VSMC, such as endothelial cells (EC), macrophages and fibroblast-like synoviocytes were sources of secreted CypA^{18, 22–23}.

eCypA has cellular functions similar to iCypA such as inflammation and proliferation, but also unique properties such as apoptosis, migration, matrix degradation and generation of ROS^{19, 24–27}. Furthermore, a receptor for eCypA, CD147, also known as Basigin or EMMPRIN, plays a role in signal transduction by eCypA²⁸. eCypA is a pro-inflammatory cytokine that promotes inflammation in many cell types. Specifically, it has a chemotactic effect on leukocytes and induces production of cytokines such as IL-1β, IL-6 and IL-8 in macrophages and monocytes^29–30. It promotes the proliferation and migration of VSMC as well as the activation of matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9 in VSMC^{19, 27, 31}. It stimulates the apoptosis of EC as well as the expression of adhesion molecules such as VCAM1 and ICAM1 in EC^24–25 (Figure 1). It enhances platelet adhesion and thrombus formation³². Activation of ERK1/2, NF-kB, Akt, JNK and p38 MAPK pathways were observed in different cell types after stimulation with eCypA^{24, 28, 33}.

Figure 1.

Cell specific effects of extracellular CypA on different cell types and the function of intracellular CypA.

The mechanisms by which eCypA mediates pro-inflammatory functions remain elusive. CD147 was demonstrated as a cell surface receptor for eCypA, and has been proposed to mediate most of its signaling³⁴. This was supported by targeting CD147 using anti-CD147 antibody caused abrogation of certain functions such as neutrophil chemotaxis in response to eCypA³⁵. The importance of eCypA interaction with CD147 has been demonstrated in several in vivo situations such as rheumatoid arthritis, pulmonary arterial hypertension, and myocardial ischemia^36–38. However, the signal transduction mechanism by which CD147 mediates eCypA signaling remains unclear. In particular, CD147 does not dimerize, increase tyrosine phosphorylation, resemble a G-protein-coupled receptor, and has no enzymatic activity (Berk unpublished data and database searches). NMR study suggested an overlap between CD147 binding site on CypA and its PPIase active site as well as the isomerization of certain peptide bonds of CD147 catalyzed by eCypA³⁹. Yurchenko et al. reported that PPIase activity of eCypA was crucial for CD147 mediated ERK1/2 activation in CD147 overexpressed CHO cells²⁸. However, it was also reported that eCypA could induce leukocyte chemotaxis via direct binding to CD147 without the involvement of its PPIase activity⁴⁰. Interestingly, it was reported that induction of IL-8 expression by eCypA in U937 cell was unaffected by knockdown of CD147 using siRNA⁴¹. In addition, Gwinn et al. reported that eosinophils expressing CD147 were not chemotactic to eCypA⁴². Furthermore, Pushkarsky et al. reported that CD147 stimulated HIV-1 infection in a signal-independent fashion⁴³. We have unpublished data that knockdown of CD147 did not affect multiple signals activated by eCypA in VSMC and EC. Therefore, the existence of CypA receptors other than CD147 that are responsible for certain activities of eCypA requires further investigation (Table 1).

Table 1.

Major extracellular CypA functions and potential signaling receptors assayed in vitro.

Effects of extracellular CypA	CD147	Unknown receptor
Apoptosis (EC)		√
Chemotaxis (leukocytes)	√
IL-8 production (U937)		√
Inflammation (EC, VSMC, etc)	√	√
MMP activation (macrophage, glial cell, etc)	√
Proliferation and migration (cancer cell, VSMC, etc)	√

Pathological roles of extracellular and intracellular Cyclophilin A in cardiovascular diseases

Vascular diseases

It is clear that eCypA contributes to the pathogenesis of vascular disease. Common pathogenic signals induced by eCypA include expression of inflammatory mediators by EC, apoptosis of EC, proliferation and migration of VSMC, and degradation of extracellular matrix due to increased MMP-2 and MMP-9 expression. Below we will discuss 5 examples (carotid intima-media thickness, coronary artery disease, peripheral artery disease, pulmonary arterial hypertension and blood-brain barrier dysfunction) where CypA plays an important role. In some examples, we can separate the role of eCypA versus total CypA while in others total CypA was studied. Future studies will be required to use eCypA specific inhibitors to show conclusively the role of eCypA.

Carotid intima-media thickness

Intimal and medial hyperplasia after carotid ligation in CypA knockout mice was significantly less than in wild type littermates. Specifically, global CypA knockout mice showed less VSMC migration and proliferation as well as less inflammatory cell accumulation in ligated arteries. Bone marrow transplantation showed that tissue resident CypA played a more important role than bone marrow derived CypA in vascular remodeling³¹. ROS was revealed as the key in vascular remodeling due to its stimulation of VSMC growth and proinflammatory effects on various cell types^44–45. We have reported that agents which stimulate oxidative stress, such as angiotensin II and LY83583 could stimulate CypA secretion from EC and VSMC while secreted CypA in turn stimulated generation of ROS in these cell types, suggesting the existence of a positive feedback loop between extracellular CypA and ROS in the pathogenesis of vascular remodeling^{25, 46}. Also mice that overexpressed CypA in VSMC showed greater eCypA by immunohistochemistry that correlated with greater intima-media thickness³¹.

Coronary artery disease (CAD)

Substantial evidence has suggested eCypA as a novel biomarker with high diagnostic and prognostic value for CAD. Yan et al. reported that plasma CypA level in patients with unstable angina (n=60) and acute myocardial infarction (n=90) were higher than those with stable angina (n=60) and normal controls (n=50), suggesting eCypA as a predictor of the severity of acute coronary syndromes, possibly due to its correlation with increased MMP-3, MMP-9 and C-reactive protein concentrations⁴⁷. Satoh et al. reported a positive correlation between plasma CypA levels and the severity of CAD. They found plasma CypA levels increased in accordance with the number of atherosclerotic risk factors, suggesting eCypA as a novel biomarker of CAD (n=320)⁴⁸. Their group also indicated that plasma CypA levels had prognostic value in patients with CAD (n=511) when combined with other traditional biomarkers such as high-sensitivity C-reactive protein and brain natriuretic peptide⁴⁹. Interestingly, a recent study by Seizer et al. showed that in patients with stable CAD (n=204), platelet-bound CypA was associated with hypertension and hypercholesterolemia. In patients with acute myocardial infarction (n=108) platelet-bound CyPA was significantly decreased.⁵⁰. In all clinical studies the significance was high (P<0.05) despite the small number of patients enrolled. These findings suggest that dynamic changes in platelet-bound CypA may have prognostic as well as pathologic importance.

Peripheral artery disease

Our group demonstrated that in mice with severe atherosclerosis due to ApoE knockout, the development of atherosclerotic aortic aneurysms in response to angiotensin II infusion was completely prevented in the CypA knockout mouse²⁷. We found both VSMC-derived iCypA and eCypA were required for the ROS generation and matrix metalloproteinase-2 activation and subsequent inflammatory cytokine expression, elastic lamina degradation, and aortic expansion. In terms of atherogenesis, Seizer et al. reported that eCypA promoted the differentiation of CD34+ progenitor cells into foam cells via CD147, thereby contributing to the progression of atherosclerosis⁵¹. Likewise, Ramachandran et al. reported that eCypA enhanced macrophage lipid uptake and foam cell formation⁵². In addition, Liu et al. reported in human patients that high plasma CypA was correlated with progressive peripheral arterial occlusion disease (n=68) and impaired renal function in chronic renal disease⁵³. Furthermore, Ramachandran et al. found higher circulating CypA level in patients with type 2 diabetes (n=313), possibly reflecting an increased oxidative stress and pro-inflammatory status compared to controls (n=122), suggesting a strong association of higher level of eCypA with diabetes and its related peripheral vascular disease⁵⁴.

Pulmonary arterial hypertension (PAH)

Oxidative stress has been implicated as a mediator of PAH. Considering the positive feedback loop between eCypA and oxidative stress, we studied the role of CypA in PAH. Our group observed a PAH phenotype in an EC specific CypA overexpression mouse model with elevated levels of eCypA and showed an essential role for eCypA in promoting pulmonary EC apoptosis, inflammation and ROS generation²⁵. Satoh et al. demonstrated high plasma CypA level in PAH patients (n=76) and CD147 knockout ameliorated experimental hypoxia-induced pulmonary hypertension in mice, indicating an essential role of eCypA in the pathogenesis of arteriole remodeling of PAH³⁷.

Blood-brain barrier dysfunction

The blood-brain barrier is formed by brain EC lining the cerebral microvasculature, and is an important mechanism for protecting the brain from harmful circulating agents such as neurotransmitters and xenobiotics⁵⁵. It has been proposed that dysfunction of the blood-brain barrier may contribute to neurodegenerative diseases such as Alzheimer’s disease⁵⁶. Several studies implicate CypA as a mediator of the ApoE4 pathway that predisposes to Alzheimer’s disease. Zlokovic and our group showed that elevated iCypA concentrations in pericytes caused nuclear translocation of NF-kB and increased activation of MMP-9⁵⁷. The intracellular CypA--NF-kB--MMP9 pathway in pericytes was associated with degradation of EC tight junctions and basement membrane, resulting in blood-brain barrier dysfunction and the extravascular accumulation of neurotoxic molecules. Furthermore, Zlokovic’s group showed that older cognitively normal APOE4 carriers (n=5) had increased cerebrospinal fluid levels of eCypA by 190% and 95% compared to cognitively normal younger APOE4 carriers (n=5) or age-matched APOE4 non-carriers (n=16), respectively ⁵⁸.

Cardiac diseases

The role of CypA in cardiac diseases has not been well studied. Our group showed that cardiac hypertrophy induced by angiotensin II in the ApoE knock out, CypA wild type mouse was attenuated in the ApoE knock out, CypA knockout mouse. This was associated with significant decreases in Ang II-induced ROS production, cardiac fibroblast proliferation and migration. Bone marrow cell transplantation showed that CypA in cells intrinsic to the heart played an important role in the cardiac hypertrophic response⁵⁹. The relative roles of eCypA versus iCypA could not be fully determined, but in this study we found eCypA directly induced protein synthesis of cultured myocytes and migration and proliferation of cultured cardiac fibroblasts, leading to cardiac hypertrophy⁵⁹. In addition, Zuern et al. has proposed that CypA is an independent predictor for clinical outcomes of patients with congestive heart failure (n=227)⁶⁰. Of all clinical, laboratory, and immunohistological parameters they tested, only CypA was identified as an independent predictor for the composite endpoint as well as for all-cause death and heart transplantation alone, suggesting the high prognostic value of CypA for these patients.

Post-translational modification of extracellular and intracellular Cyclophilin A

Both extracellular and intracellular CypA undergo post-translational modification. In HEK293 cells, serine/threonine residues of iCypA undergo phosphorylation in response to activation of chemokine receptor CXCR4⁶¹. Phosphorylated CypA may then play a role in CXCR4 signaling. Glutathionylation of iCypA is observed in T cells and hepatocytes under oxidative stress⁶². Recently, it has become clear that oxidative stress promotes the acetylation of CypA⁴⁶. Several lysine residues are critical for acetylation of CypA and its biological functions^63–67 (Table 2). A correlation between phosphorylated and acetylated CypA and oxidative stress is indicated by proteomic analysis of spinal cord homogenates from presymptomatic amyotrophic lateral sclerosis G93A SOD1 mice⁶⁸. Angiotensin II induced iCypA acetylation and acetylated CypA secretion in VSMC⁴⁶. Importantly, substantial evidence indicates that extracellular acetylated CypA is a more potent agonist for EC and VSMC activation than non-acetylated CypA²⁵. In addition, acetylation has been shown to interfere with the binding ability of iCypA to HIV-1 caspids, thus reducing the viral infectivity⁶³. The emerging critical role of extracellular and intracellular acetylated CypA in disease pathology makes it a novel target for therapy.

Table 2.

Critical lysine residues and their biological functions in acetylation of CypA.

Lysine residues	Conservation	Location	Function when not Acetylated	Function when Acetylated
K28	Not conserved	Inside
K44	Conserved in all species	Inside
K82	Conserved in most species	Surface	Calcineurin and CsA-CypA complex binding	1. EC apoptosis 2. EC inflammation 3. VSMC proliferation
K125	Conserved in most species	Surface	Calcineurin and CsA-CypA complex binding	1. PPIase inhibition 2. CsA-CypA complex binding inhibition 3. EC apoptosis 4. EC inflammation 5. VSMC proliferation 6. HIV-1-CypA interaction alteration
K131	Not conserved	Inside

Drugs that block extracellular and intracellular Cyclophilin A

Several drugs have been developed to study the function of CypA; most of these inhibit CypA PPIase activity. CsA is a cell permeable compound that is the best studied inhibitor of CypA PPIase activity. The binding of CsA to iCypA inhibits calcineurin and prevents the translocation of transcription factor nuclear factor of activated T-cells into the nucleus, thereby inhibiting the production of cytokines⁵. CsA is now broadly used as an immunosuppressant due to its inhibitory effect upon T cell responses. However, its potent immunosuppressive activity prevents CsA from being widely used in the treatment of cardiovascular and inflammatory diseases. Because of this, non-immunosuppressive CsA derivatives such as N-methyl-4-isoleucine cyclosporin (NIM811) and alisporivir were developed. It was reported that both NIM811 and alisporivir inhibited viral replication of HIV-1 and HCV^69–72. Seizer et al. reported that NIM811 inhibited myocardial fibrosis and inflammatory lesions in murine coxsackievirus B3-induced myocarditis⁷³. Stemmy et al. reported a reduction of leukocyte influx into the airways in a murine model of chronic asthma after application of NIM811⁷⁴. Recently, Ahmed-Belkacema et al. reported a fragment-based selection approach to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors, unrelated to CsA, with potent in vitro PPIase inhibitory activity and antiviral activity against HIV and HCV⁷⁵.

As mentioned above, plasma levels of CypA have been found to correlate with disease progression and severity in many diseases. These findings suggest that eCypA may be more important than iCypA as a pathogenic mediator of disease. Because CsA and derivatives such as NIM811 inhibit both extracellular and intracellular CypA, drugs have been developed to study the function of eCypA. Specifically, scientists have modified the chemical groups of CsA that affect permeability, making the analogs impermeable. Due to this cell-impermeable property, the new derivatives can selectively inhibit eCypA without interfering the normal physiological functions of iCypA. MM218, as one of the recently synthesized cell-impermeable CsA derivatives, contains a highly charged moiety that prevents its passage through the plasma membrane, thereby making it a specific eCypA inhibitor⁷⁶. Balsley et al. showed that MM218 effectively reduced inflammatory responses in a mouse model of allergic lung inflammation by blocking leukocyte recruitment mediated by extracellular cyclophilins⁷⁷. Pasetto et al. reported MM218 rescued motor neurons and extended survival of an ALS mouse model by blocking eCypA⁷⁸. MM284 is another selective eCypA inhibitor similar to MM218. It has been reported that MM284 reduced myocardial Inflammation and remodeling in a mouse model of myocarditis⁷⁹. Seizer et al. showed that ADP-induced platelet aggregation was attenuated by MM284. A recent study indicated that MM284 inhibited SMAD activation and inflammation in an experimental biliary atresia model, ameliorating disease symptoms⁸⁰.

Clinical relevance of extracellular CypA

The inflammatory hypothesis as a medidator of atheroscletosis received strong support from the recent Canakinumab Antiinflammatory Thrombosis Outcome Study conducted by Ridker et al.. They used a monoclonal antibody targeting interleukin-1β in patients with CAD (n=10061) and showed a significantly lower rate of recurrent cardiovascular events over a median of 3.7 years⁸¹. There are many similarities in the pro-inflammatory effects of extracellular interleukin-1β and extracellular CypA on cells in the cardiovascular system. Our lab has identified eCypA as a pathological mediator of atherosclerosis, aneurysm and PAH due to its pro-inflammatory effects^{27, 31, 82}. The positive results of antibody to interleukin-1β suggest that preclinical data from the CypA relevant animal models mentioned above may have relevance to human cardiovascular diseases (Table 3). A clinical trial by Cung et al. tested the effect of CsA on clinical outcome and left ventricular remodeling in patients undergoing percutaneous coronary intervention for an acute myocardial infarction within 12 hours (n=970). There was no benefit of CsA⁸³. It should be noted that CsA may not be a good drug because of its broad immunosuppressive effects and its inhibition of iCypA as well as eCypA. Future studies will be necessary to determine the prognostic value of circulating eCypA and post-translational modified CypA for different cardiovascular diseases. Also, PPIase inhibitors targeting eCypA such as MM284 may be effective in high risk populations like the Canakinumab Antiinflammatory Thrombosis Outcome Study population, or in the acute myocardial infarction setting due to its effects on platelets, immune cells and cells of the vessel wall. In addition, CypA may potentially be an excellent marker because it could be a pathogenic marker both causative and predictive of disease. This is possible because it represents the combination of multiple atherosclerotic mechanisms. Specifically, it combines ROS, inflammation, and matrix degradation. It also involves paracrine stimulation as a secreted protein and modification by acetylation appears to make it a more potent mediator, potentially representing a metabolomic indicator. Understanding the “life cycle” of CypA during cardiovascular disease will be necessary to find how to use it as a biomarker.

Table 3. Cardiovascular diseases with clear evidence that extracellular CypA plays a role in pathogenesis.

The only human data are for coronary artery disease, peripheral arterial occulusion and pulmonary arterial hypertension where plasma CypA correlated with disease progression and/or severity; and hypertension and hypercholesterolemia where platelet-bound CypA correlated with. Other studies are in mouse. The right column stands for disease in which total CypA is studied, thus we can not tell the effects of eCypA from iCypA.

Cardiovascular Diseases	eCypA	Total CypA	References
Carotid intima-media thickness		√	31, 44, 45
Coronary artery disease	√		47, 48, 49
Hypertension	√		50
Hypercholesterolemia	√		50
Atherosclerotic aortic aneurysm		√	27
Atherosclerosis	√		51, 52
Peripheral arterial occlusion	√		53
Pulmonary arterial hypertension	√		25, 37
Cardiac hypertrophy		√	59

Summary

Both extracellular and intracellular CypA have been shown to play pathological roles in animal models of many diseases. Recent data in humans show that the level of plasma CypA correlates with disease progression and severity. Cardiovascular diseases in which CypA plays a potential pathogenic role include carotid intima-media thickness, coronary artery disease, peripheral artery disease, pulmonary artery hypertension and blood-brain barrier dysfunction. This diversity of diseases supports the concept that extracellular CypA is a pathogenic mediator of cardiovascular disease. This concept is further strengthened by promising results with extracellular CypA inhibitors in several mouse models of human disease. Based on the evolving pathogenic role of circulating CypA, drugs that specifically inhibit extracellular CypA may be effective in multiple diseases.

Highlights.

1. Both extracellular and intracellular CypA play important roles in cardiovascular disease pathogenesis through shared and unique mechanisms.

2. Substantial evidence reveals extracellular CypA as a potent mediator of cardiovascular diseases through a specific receptor-(CD147 or another) mediated inflammation, matrix degradation and ROS generation.

3. Post-translational modification of extracellular and intracellular CypA, especially acetylation, may strongly influence their biological functions.

4. Increasing evidence shows a correlation between circulating extracellular CypA and cardiovascular disease severity, suggesting that extracellular CypA may not only be a good biomarker, but potentially a good therapeutic target for drug development.

Abbreviations

CypA

cyclophilin A

PPIase

peptidyl prolyl isomerase

CsA

cyclosporin A

eCypA

extracellular cyclophilin A

iCypA

intracellular cyclophilin A

ROS

reactive oxygen species

VSMC

vascular smooth muscle cell

EC

endothelial cell

MMP

matrix matrix metalloproteinase

CAD

coronary artery disease

PAH

pulmonary arterial hypertension