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Cyclophilin A (CyPA) has been studied as a multifunctional protein that is upregulated in a variety of inflammatory conditions, such as rheumatoid arthritis, autoimmune disease, and cancer. CyPA has been classified as an immunophilin and has a variety of intracellular functions, including intracellular signaling, protein trafficking, and the regulation of other proteins activity. Besides its intracellular functions, CyPA is a secreted molecule that has a physiological and pathological role in cardiovascular diseases, making it a potential biomarker and mediator in cardiovascular diseases, such as vascular stenosis, atherosclerosis, and abdominal aortic aneurysms.
The interaction between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular integrity. ECs secrete a variety of vasoactive substances, including nitric oxide (NO) and prostacyclin, which protect against vascular remodeling.1,2 VSMCs contain numerous sources of reactive oxygen species (ROS; ie, H2O2, O2−, and ·OH), including NADPH oxidases, xanthine oxidase, the mitochondrial respiratory chain, lipoxygenases and NO synthases.
Oxidative stress, generated by excessive ROS, promotes cardiovascular disease. However, the precise mechanism of the deterioration in vascular function and promotion of vascular remodeling by ROS in vivo has not been clearly elucidated. VSMCs are among the most plastic of all cells in their ability to respond to different stimuli. Autocrine/paracrine growth factors from VSMCs have been mentioned for a long time as important mechanisms that mediate the varying cellular responses in vascular remodeling.3–5 It has now become clear that almost all VSMC growth factors elicit auto/paracrine growth pathways. Recent evidence suggests that many other stimuli that modulate VSMC function, including ROS, promote VSMC growth by inducing auto/paracrine growth mechanisms (as reviewed by Taniyama and Griendling6). ROS increase cell proliferation, mediate agonist-induced hypertrophy, and also induce apoptosis in a concentration-dependent manner.7
Cyclophilin A (CyPA) is a 20-kD chaperone protein secreted from VSMCs in response to ROS, and it stimulates VSMC proliferation and inflammatory cell migration in vitro and in vivo. The major topics that will be addressed in this review are a series of projects that were performed in our laboratory. Does ROS-induced secreted CyPA actually contribute to several cardiovascular diseases in vivo? To answer this question, we used several genetic interventions, including the CyPA knockout mouse and the CyPA overexpressing transgenic mouse (VSMC-Tg). Thus, we elucidated that CyPA mediates a variety of cardiovascular diseases, including vascular stenosis, atherosclerosis, and abdominal aortic aneurysm (AAA). In this review we will discuss these recently revealed roles of CyPA in cardiovascular disease.
In 1984, CyPA was identified as the main target for the immunosuppressive drug cyclosporine A (CsA).8–11 Cyclophilins are a family of highly conserved and ubiquitous proteins, termed immunophilins.12 The most abundant cyclophilin is CyPA,13 which is widely distributed in almost all tissues in prokaryotes and eukaryotes. In humans, CyPA has been found in all organs and the CyPA concentration may account for as much as 0.1–0.4% of the total protein in a cell.14–16 CyPA is abundant in the cytosolic extract from lymphocytes and has a high affinity for CsA.8 CyPA was also shown to be a part of a cytosolic heat-shock protein – immunophilin chaperone complex that includes caveolin and cholesterol.17 Because of its enzymatic properties, cellular localization, and role in protein folding, CyPA belongs to a diverse set of proteins known as molecular chaperones. Because CyPA catalyzes the cis-trans isomerization of the peptidyl-prolyl bonds of certain proteins (PPIase activity), CyPA acts as acceleration factor in protein folding and assembly. The first demonstration of this activity in vitro was delaying the maturation of collagen by blocking PPIase activity with CsA.18 In addition to its role in protein folding, the PPIase activity of CyPA has recently been demonstrated to have other roles, including intracellular trafficking,19 signal transduction, and transcription regulation.20 Following the identification of CyPA, several other cyclophilins were cloned and characterized. Cyclophilin B (CyPB),21 cyclophilin C (CyPC),22 and cyclophilin D (CyPD)23 were found to be less abundant and localized not only in the cytosol but also in membranes and subcellular organelles because of the presence of a hydrophobic N-terminal as well as C-terminal extensions. Human CyPB and murine CyPC are localized to the endoplasmic reticulum.23 CyPD is localized to mitochondria and is an integral part of the mitochondrial permeability transition complex and plays a crucial role in apoptosis24 and the pathogenesis of Alzheimer’s disease.25 A more detailed classification of the different cyclophilins has been reviewed recently.13,26
Production of intracellular ROS has been implicated in the pathogenesis of cardiovascular disease, in part by the promoting of VSMC proliferation.27–29 Changes in vascular redox state are a common pathway involved in the pathogenesis of atherosclerosis, aortic aneurysms, and vascular restenosis after angioplasty. ROS target cellular biomolecules and cause severe damage, such as lipid peroxidation, protein oxidation/inactivation, and DNA damage/mutation. Although high levels of ROS might be hazardous to cells and their contents, controlled ROS levels (ie, physiological) are important in the regulation of cell functions and cell fate (proliferation/death). For example, H2O2 has also been implicated as important for EC function and vascular relaxation at very low concentrations.30,31 In the vascular wall, ROS are generated by several mechanisms, including NADPH oxidases, xanthine oxidase, the mitochondrial respiratory chain, lipoxygenases and NO synthases.32 Vascular ROS formation can be stimulated by mechanical stretch, pressure, shear stress, environmental factors such as hypoxia, and secreted factors such as angiotensin II (AngII).33 We have demonstrated that ROS stimulate cultured VSMC proliferation and activate intracellular kinases such as ERK1/2 which is associated with cell growth.29,34
We found that activation of ERK1/2 by a ROS generator, napthoquinolinedione (LY83583), was biphasic (early and delayed activation). One explanation for the delayed ERK1/2 activation was the response to SOXF, which show autocrine/paracrine signals. In order to identify the presence of SOXF, we evaluated the ability of conditioned medium for ERK1/2 activation. The phosphorylation of ERK1/2 was significantly increased by conditioned medium from VSMCs treated with LY83583. Therefore, we analyzed the proteins released into the medium in response to LY83583 and finally found that CyPA is a major SOXF.35 Furthermore, human recombinant CyPA stimulated ERK1/2 activity and DNA synthesis in VSMCs in a concentration-dependent manner.36 Thus, we concluded that CyPA is a novel VSMC growth factor that contributes to the growth promoting activity of ROS in VSMCs.
Identification of the extracellular CyPA receptors is almost completely unexplored. We believe that further knowledge of the role played by extracellular CyPA receptors on vascular cell responses will help in designing therapeutics targeting inflammatory and cardiovascular diseases. In ECs, CyPA largely activates proinflammatory pathways, including increased expression of vascular cell adhesion molecule (VCAM)-1and E-selectin.37 In VSMCs, ROS such as superoxide activate a pathway containing vesicles that results in secretion of CyPA.38 Secreted extracellular CyPA stimulates ERK1/2, Akt and JAK in VSMCs, which contributes to ROS production again (Figure 1).36 Despite the mounting evidence that cyclophilins serve multiple intracellular and extracellular functions, surprisingly little is known regarding their effect on specific receptors. Several molecules have been proposed as potential extracellular receptors for CyPA, including extracellular matrix metalloproteinase inducer (EMMPRIN).39,40
It has been revealed that several growth factors are secreted from VSMCs in response to various stimuli.41 CyPA is secreted from VSMCs via a highly regulated pathway that involves vesicle transport and plasma membrane binding.38 Rho GTPases, including RhoA, Cdc42, and Rac1, are key regulators in signaling pathways linked to actin cytoskeletal rearrangement.42 The Rho GTPases plays a central role in vesicular trafficking pathways by controlling the organization of the actin cytoskeleton. It has been reported that active participation of Rho GTPases is required for secretion of CyPA. We have shown consistently that the expression of dominant-negative mutants of RhoA and Cdc42 inhibited ROS-induced CyPA secretion, suggesting that both RhoA-and Cdc42-dependent signaling events regulate CyPA secretion.38 Myosin II is involved in secretory mechanisms as a motor for vesicle transport.43 Rho-kinase, a downstream effector of RhoA, mediates myosin II activation via phosphorylation and inactivation of myosin II light chain phosphatase.44 We also demonstrated that a Rho-kinase inhibitor reduced ROS-induced CyPA secretion.38,45 These results suggest that myosin II-mediated vesicle transport is required for CyPA secretion from VSMCs. CyPA is transported to the plasma membrane and colocalizes with VAMP in response to ROS stimulation. Therefore, CyPA is secreted from VSMCs through a process requiring ROS production and vesicle formation.
Increases in ROS represent a pathogenic mechanism for vascular disease.46,47 ROS have been implicated in the pathogenesis of neointima formation, in part by promoting VSMC growth,29,34 as well as by stimulating proinflammatory events.48–51 We demonstrated that extracellular CyPA stimulates proinflammatory signals in ECs, including expression of E-selectin and VCAM-1.37 In addition to the effects on vascular cells, CyPA has been shown to be a direct chemoattractant for inflammatory cells52,53 and to promote matrix metalloproteinases (MMPs) activation.54,55 Therefore, CyPA is a key mediator that affects ECs, VSMCs and inflammatory cell functions in vivo.
To confirm the role of CyPA in vascular remodeling, we observed the time course and distribution of its expression in carotid arteries after ligation.56 We found that CyPA expression dramatically increased over a time course that paralleled neointimal formation, suggesting an important role for CyPA in the cellular response to oxidative stress induced by vascular injury. In parallel with CyPA expression, carotid ligation induced phosphorylation of ERK1/2 in wild-type carotids, which was significantly less in CyPA−/− carotids, consistent with the reduced number of Ki67+ cells in ligated CyPA−/− carotids. The distribution of Ki67+ cells closely overlapped with the areas of highest CyPA expression, especially in rapidly proliferating neointimal cells in WT mice. Colocalization of CyPA, α-smooth muscle actin (αSMA), and Masson-Trichrome staining revealed that CyPA expression was especially elevated in VSMCs. To prove further the contribution of VSMC-derived CyPA to vascular remodeling, we prepared VSMC-specific CyPA transgenic mice (VSMC-Tg). The observation that VSMC-specific CyPA overexpression not only increased the medial area but also the intimal area suggests that VSMC-derived extracellular CyPA promotes the proliferation and migration of VSMCs via a paracrine manner. CyPA is expressed by all cell types participating in vascular pathology.57 Additionally, extracellular CyPA has recently been found to induce interleukin (IL)-6 release in inflammatory cells.58 Moreover, investigating CyPA function in monocyte/macrophage cell lines revealed that CyPA induces the expression of cytokines/chemokines such as tumor-necrosis factor α, monocyte chemotactic protein-1, IL-8, IL-1β and MMP-9 through a pathway that is dependent on nuclear factor-κB activation. In our carotid ligation model, we observed significant accumulation of CD45+ inflammatory cells in the intima of ligated CyPA−/− carotids and the VSMC-specific overexpression of CyPA (VSMC-Tg) further enhanced the accumulation of inflammatory cells in the ligated carotids, supporting the important role of CyPA in mediating the recruitment of inflammatory cells (Figure 2).56
We propose that ROS generated locally by inflammatory cells cause VSMCs to release CyPA, which would then promote recruitment of inflammatory cells that release several proinflammatory cytokines. In addition, CyPA regulates the proteolytic activity necessary for the migration of inflammatory cells, through its activation of MMPs. Our study revealed 3 important pathologic consequences of CyPA activity in vivo. First, VSMC-derived secreted CyPA is mitogenic by virtue of its ability to promote VSMC proliferation. Second, secreted extracellular CyPA is proinflammatory because it stimulates the recruitment of inflammatory cells. Third, secreted CyPA promoted the pathological setting that exacerbated the generation of intracellular ROS in VSMC derived from mouse aorta (Figure 2).
As we have discussed, ROS stimulate secretion of CyPA from VSMCs, and that extracellular CyPA stimulates VSMC migration and proliferation (Figure 1).35,36 Extracellular CyPA also stimulates EC adhesion molecule expression, and is a chemoattractant for inflammatory cells.36,38,59 Furthermore, CyPA is upregulated in patients with rheumatoid arthritis and implicated because of its crucial role in MMP activation.52 AngII infusion into ApoE−/− mice for 4 weeks promotes AAA formation.60,61 In animal models of AAA, genetic and pharmacological inhibition of both ROS production62,63 and MMPs64,65 suppressed development of aneurysms. In that animal model, the AngII type 1 (AT1) receptor in the vascular wall, but not in inflammatory cells, is required for the initiation of AngII-induced AAAs.66 Furthermore, treatment with an AT1 receptor blocker significantly suppressed aneurysm formation in ApoE−/− mice.67 Therefore, we hypothesize that VSMC-derived CyPA augments AngII-induced ROS production, MMP activation, and inflammatory cell recruitment into the aortic VSMCs, contributing to AAA formation and progression.
In the cardiovascular system, AAA formation results from chronic inflammation of the aortic wall, associated with decreased medial VSMCs, and progressive destruction of structural components, particularly the elastic lamina.68 Key mechanisms include VSMC senescence,69 oxidative stress,6,46 increased local production of proinflammatory cytokines70 and increased activities of MMPs that degrade extracellular matrix.71,72 As expected, AAA formation in the AngII-induced ApoE−/− model was completely prevented against a CyPA−/− background.45 We also demonstrated that CyPA is highly expressed in the aorta of patients with AAA, and colocalizes with active forms of MMPs. Based on these findings, we demonstrated that AngII induces ROS and MMP activation via a CyPA-dependent pathway, a novel mechanism for induction of AAA formation by AngII.
Our data suggest that extracellular CyPA and its signaling pathways are novel targets for AAA therapy and, potentially, other cardiovascular diseases associated with inflammation. In addition, extracellular CyPA induces ROS production in VSMCs, which is consistent with our previous report that extracellular CyPA stimulates at least 3 signaling pathways (ERK1/2, Akt and JAK) in VSMCs,36 which has been shown to be important for ROS production.6,46 All these data are proof-of-concept that CyPA plays a crucial role in VSMCs through ROS generation. AngII induces the generation of ROS and promotes the secretion of CyPA. ROS-induced CyPA secretion augments ROS production synergistically (Figure 1). Subsequently, secreted CyPA, acting as a proinflammatory cytokine, synergistically augments AngII-mediated ROS production, contributing to the onset of vascular inflammatory cell migration and AAA formation (Figure 3).62
Numerous basic and clinical studies have clearly identified that ROS have a major role in endothelial damage and the development of atherosclerosis.73–75 However, we still do not have a strong therapeutic strategy for the clinical benefits of antioxidant administration. One potential reason for this could be the crucial role of ROS (especially H2O2) at very low concentration in intracellular signaling pathways that are also important for vascular functions.30,31,76,77 CyPA (both intracellular and extracellular) contributes to atherosclerosis by promoting EC apoptosis and EC expression of leukocyte adhesion molecules, stimulating inflammatory cell migration, enhancing ROS production, increasing proliferation of macrophages and VSMCs, and increasing proinflammatory signal transduction in VSMCs.78,79 In the context of atherosclerosis, CyPA can be regarded as a proinflammatory and proatherogenic molecule. CyPA is highly expressed at sites of unstable atherosclerotic plaques, especially those associated with macrophages and foam cells. However, CyPA expression and its regulatory molecular mechanisms during the process of plaque destabilization remain elusive and further research into the role of CyPA in the progression of atherosclerosis is needed to identify potential CyPA-related therapeutic targets.
AngII plays a key role in many physiological and pathological processes in cardiac cells, including cardiac hypertrophy.80 Therefore, understanding the molecular mechanisms responsible for AngII-mediated myocardial pathophysiology is critical to the development of new therapies for cardiac dysfunction.81 One important mechanism now recognized as involved in AngII-induced cardiac hypertrophy is ROS production, 82,83 but the precise mechanism by which ROS cause hypertrophy remains unknown.84 Our recent study provides strong mechanistic evidence of synergy between CyPA and AngII to increase ROS generation.45 Because ROS stimulate myocardial hypertrophy, matrix remodeling, and cellular dysfunction,85 CyPA will potentially enhance AngII-induced cardiac hypertrophy.
PAH is associated with hypoxic exposure, enhanced ROS, and proliferation of VSMCs. Erythropoietin (Epo) has long been regarded as a hypoxia-induced hormone that acts exclusively in the proliferation and differentiation of erythroid progenitors. However, recent studies have demonstrated expression of the Epo receptor (EpoR) in the cardiovascular system,86 and the therapeutic potential of Epo has been noted in a variety of disorders, including cerebral infarction, myocardial ischemia – reperfusion, and congestive heart failure. Recently, we demonstrated that the endogenous Epo/EpoR system plays an important protective role against the development of hypoxia-induced PAH.87 For this purpose, we used EpoR−/−-rescued mice that express EpoR only in the erythroid lineage, but not in th cardiovascular system.86 Moreover, we demonstrated the important role of the endogenous Epo/EpoR system in ischemia-induced regeneration and angiogenesis.88
Considering the role of CyPA in augmentation of ROS and VSMC proliferation and migration in vivo and in vitro, CyPA may potentially promote hypoxia-induced PAH. We have reported that Rho-kinase is activated in patients with PAH.89 In addition, the secretion of CyPA is regulated by the Rho/Rho-kinase system.38,45 Therefore, we tested the hypothesis that CyPA contributes to Rho-kinase activation and pulmonary vascular remodeling in PAH patients.90 A key aspect of the study that deserves comment is the strong CyPA expression on αSMA-positive cells in the lungs of patients with idiopathic PAH. It is reported that bone marrow-derived αSMA-positive cells contribute to the development of PAH91 and promote atherosclerotic plaque stability.92 Additionally, we have reported that statins and a Rho-kinase inhibitor reduced the secretion of CyPA from VSMCs,38,45 and demonstrated that pravastatin ameliorates hypoxia-induced PAH in mice.93 Based on this, inhibition of CyPA secretion by statins93 or Rho-kinase inhibitor94,95 may contribute to the therapeutic effect of these drugs in PAH patients.
The identification of CyPA as a mediator of tissue damage associated with inflammation and oxidative stress provides insight into the mechanisms of several therapies. For example, the Rho-kinase inhibitor, Y27632, and simvastatin significantly reduced CyPA secretion from VSMCs. Rho-kinase is an important therapeutic target in cardiovascular disease96 and Rho-kinase inhibition has been reported to reduce AngII-induced AAA formation,97 atherosclerosis, and cardiac hypertrophy.98 Moreover, AT1a receptor blockers and angiotensin-converting enzyme inhibitors have been shown to prevent cardiovascular diseases,66,67,99 and reduced CyPA secretion may partially contribute to the therapeutic effect of these drugs on AAA, atherosclerosis, and cardiac hypertrophy.45 EMMPRIN, a putative CyPA receptor, was identified as a tumor cell membrane protein that is expressed in VSMCs, activated by ROS and stimulates MMP production.100 A recent study demonstrated ROS-dependent increases in EMMPRIN,101 which may be activated by binding of extracellular CyPA.102 Moreover, it has been demonstrated that EMMPRIN is strongly expressed in human AAA lesions103 and in cardiomyocytes.104 Therefore, it is logical to propose that agents that prevent CyPA binding to its receptors may have therapeutic potential (Figure 1). Because inflammation and oxidative stress contribute to tissue damage in several situations, such as ischemia – reperfusion injury in the brain, heart and kidney, future studies of CyPA-mediated function in appropriate models may reveal its significant role in other diseases. By blocking the vicious cycle that augments ROS production through the CyPA autocrine/paracrine signaling pathway, we may have a novel therapeutic tool for controlling cardiovascular diseases in the near future.
This work was supported by NIH grant HL49192 (B.C. Berk), Astra-Zeneca Research Grant (K. Satoh), grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan (K. Satoh), and Grants-in-aid for scientific research from the Ministry of Health, Labour, and Welfare, Tokyo, Japan. We are grateful to members of the Berk lab in the Aab Cardiovascular Research Institute at the University of Rochester School of Medicine for useful suggestions, especially the work performed by Duan-Fang Liao, Zheng-Gen Jin, Jun Suzuki, Tetsuya Matoba, and Patrizia Nigro.