Abstract
Chronic vascular diseases such as atherosclerosis, aneurysms, diabetic angiopathy/retinopathy as well as fibrotic and proliferative vascular diseases are generally complicated by the progression of degenerative insults, which are characterized by endothelial dysfunction, apoptotic/necrotic cell death in vascular/immune cells, remodeling of extracellular matrix or breakdown of elastic lamella. Increasing evidence suggests that dysfunctional calpain proteolytic systems and defective calpain protein metabolism in blood vessels contribute to degenerative disorders. In vascular endothelial cells, the overactivation of conventional calpains consisting of calpain-1 and -2 isozymes can lead to the disorganization of cell-cell junctions, dysfunction of nitric oxide synthase, sensitization of Janus kinase/signal transducer and activator of transcription cascades and depletion of prostaglandin I2, which contributes to degenerative disorders. In addition to endothelial cell dysfunctions, calpain overactivation results in inflammatory insults in macrophages and excessive fibrogenic/proliferative signaling in vascular smooth muscle cells. Moreover, calpain-6, a non-proteolytic unconventional calpain, is involved in the conversion of macrophages to a pro-atherogenic phenotype, leading to the pinocytotic deposition of low-density lipoprotein cholesterol in the cells. Here, we discuss the recent progress that has been made in our understanding of how calpain contributes to degenerative vascular disorders.
Keywords: Calpastatin, Vascular inflammation, Pathological angiogenesis, Extracellular matrix, Oxidative stress
Introduction
Majority chronic vascular diseases, including atherosclerosis, aneurysms, diabetic angiopathy and proliferative retinopathy as well as fibrotic vascular diseases, harbor unique and common degenerative insults1–6). For example, atherosclerotic vessels frequently accompany dysfunction of the vascular endothelial cells (ECs), accumulation of cholesterol-enriched cellular apoptotic bodies, remodeling of the extracellular matrix (ECM) and breakdown of elastic lamella2). Although the deposition of cholesterol is unique to atherosclerotic diseases, the endothelial dysfunction and ECM remodeling are frequently detected in other vascular diseases, such as diabetic angiopathy and vascular fibrosis. In general, vascular degenerations are driven by chronic inflammatory responses and accompanying reactive oxygen species (ROS)-mediated oxidative stress7); thus, many researchers have tested anti-oxidative agents in clinical trials. Unfortunately, these antioxidative trials have thoroughly failed to reverse chronic vascular diseases8), although they were effective in acute inflammation, such as the acute phase of stroke9). Therefore, it is necessary to investigate how blood vessels shift toward degenerative status in chronic disease. In this regard, growing evidence suggests that the calpain proteolytic systems, comprised of Ca2+-dependent intracellular proteases and a calpain endogenous inhibitor, critically contribute to degenerative vascular disorders. Calpains can be potentiated in the presence of pathophysiological stressors, such as inflammatory cytokines, growth factors, bioactive lipids and hypoxic stimulus10, 11). As a result, dysregulation of these systems might be responsible for the vascular disorders noted above. In this review article, we discuss the current achievements in understanding the pathophysiology of degenerative vascular disorders, with a particular focus on the calpain proteolytic system.
Vascular Calpain Systems
Calpains comprised of 15 homologues in mammals can be categorized as conventional and unconventional isozymes. Conventional calpains consist of two ubiquitous isozymes, calpain-1 and -2, which require micromolar and millimolar levels of Ca2+ for half-maximal activation, respectively12–14). These ubiquitous isozymes are comprised of heterodimers containing a common regulatory subunit, CAPNS1, and subtype-specific large subunits, CAPN1 and CAPN2, which function as the catalytic subunits of calpain-1 and -2 isozymes, respectively12–14) (Fig. 1). Conventional calpains are localized to the majority of vascular systems, including veins15), arteries16) and capillaries17), and are also expressed in ECs16, 18), vascular smooth muscle cells (VSMCs)19) and adventitial fibroblasts20). In addition to the vascular component cells, calpains are localized to immune cells21, 22), which can be recruited into blood vessels in response to inflammatory insults. In addition to the conventional calpain isozymes, calpastatin, an endogenous calpain inhibitor, colocalizes with the proteases, and negatively regulates their proteolytic activity12–14). It was reported that deficiency of the Capn1 gene in mice resulted in normal growth with a reduction in platelet aggregation and clot retraction23); therefore, calpain-1 does not play a vital role in these functions. In contrast, Capn2-deficient mice showed embryonic lethality between the morula and blastocyst stages24). Similar to Capn2, deficiency of Capns1 was embryonic lethal and accompanied by disorders in cardiovascular development25). Takano et al. subsequently reported that conditional knockout mice, in which Capn2 was expressed in the placenta but not in the fetus, survived to adulthood26). Thus, calpain-2 is unlikely to play an essential role in cardiovascular development in the fetus.
Fig. 1.
Vascular calpain systems. Conventional calpains are localized to the majority of the vascular component cells, including vascular endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and fibroblasts. Calpain-6, a non-proteolytic unconventional calpain, is inducible in foamy macrophages in athero-prone vessels. Calpastatin, an endogenous inhibitor, colocalizes with conventional isozymes, and downregulates their proteolytic activity. PC: protease core domain, CBSW: calpain-type β-sandwich domain, PEF: penta-EF-hand domain, GR: glycinerich domain, C2: C2 domain, XL: XL domain, L: L domain.
In general, physiological substrates of conventional calpains are hard to predict, because the proteases do not strictly recognize unique consensus amino acid sequences or structural motifs in their substrates. Earlier enzymatic investigations reported that conventional calpains post-translationally proteolyze a variety of substrates, including the focal adhesion proteins talin27) and vinculin28), leading to alterations in cell motility and morphology. Furthermore, the calpain-dependent post-translational regulation of signaling molecules such as focal adhesion kinases29), protein kinase C30) and inhibitor of κB (IκB)31, 32) have been described previously. It was documented that conventional calpains proteolytically degrade the EC-derived angiostatic peptide vasohibin-1 in tumor cells33). Recently, we identified VE-cadherin, an inter-endothelial adhesion molecule34), and suppressor of cytokine signaling 3 (SOCS3), an endogenous inhibitor of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cytokine signaling35) as substrates of the calpain system. These data suggest that conventional calpains recognize diverse substrates and modify multiple signaling cascades in cells, and it is suspected that their impact on cellular functions is dependent upon cell type or environmental factors, such as the type of inducer. Please refer to the following excellent reviews for extensive reviews of the molecular basis of conventional calpains12–14).
In addition to conventional isozymes, 13 species of calpain catalytic subunit have been identified in mammals. These unconventional isoforms are further categorized into ubiquitous and tissue-specific forms according to their tissue localization in vertebrates12–14). Compared with the conventional isoforms, the roles of the unconventional isoforms in vascular regulation have been less extensively investigated, probably because of their low expression in the vasculature. Indeed, our preliminary data showed that the expression levels of unconventional calpains in murine normal healthy aorta are much lower in comparison with those of Capn1, Capn2, Capns1 and Cast (our unpublished observations); nevertheless, it is suspected that certain unconventional isozymes can be potentiated under pathophysiological conditions. For instance, calpain-6 is induced in foamy macrophages in response to inflammatory stimuli during atherogenesis36), whereas this molecule was reportedly localized to fetal skeletal muscle, cartilage and heart37) as well as the placenta in adults38) but not to blood vessels during normal physiological status. This subtype appears to lack proteolytic activity, as the cysteine residue in its active core is substituted with lysine12–14) (Fig. 1). Tonami et al. identified that the deletion of Capn6 in mice promoted the development of embryonic skeletal muscle37). Furthermore, calpain-6 was induced during the regeneration of skeletal muscles in adult mice after cardiotoxin-induced degeneration, and Capn6 deficiency accelerated skeletal-muscle regeneration in mice37); however, the role of calpain-6 in the physiological regulation of vascular systems is currently poorly understood.
Calpain-10 is ubiquitously expressed and is associated with apoptosis in pancreatic islet cells39), mitochondrial dysfunction40), insulin secretion from pancreatic islets41, 42), and the oxidative utilization of glucose in skeletal muscle43). Capn10 deficiency in the SM/J mouse strain ameliorated insulin resistance and reduced blood glucose levels44). Furthermore, a genome-wide analysis has shown that a CAPN10 polymorphism is involved in insulin resistance, dyslipidemia, and high free fatty acid levels in a Japanese population45); obesity in a Scandinavian population46); and free fatty acid levels in a Finnish population47). It is likely that genetic variation in the CAPN10 locus confers higher cardiovascular disease risk to type 2 diabetes mellitus patients48). Therefore, calpain-10 might induce diabetic angiopathy through its diabetogenic actions, whereas the direct role of this molecule in vascular regulation is unknown.
Contribution of Calpain Proteolytic Systems to Degenerative Vascular Disorders
Atherosclerosis and Aneurysmal Diseases
Atherosclerosis is a vascular disease characterized by the intimal thickening of systemic arteries, including the coronary and cerebral arteries as well as the aorta49, 50). Vulnerable and occlusive atherosclerotic plaques can lead to lethal cardiovascular events, including myocardial infarction and stroke, two primary causes of morbidity and mortality worldwide. Because many pathogenic cues contribute to atherogenesis50), it is difficult to precisely define the cause of atherosclerosis; however, the majority of investigations are based on the hypothesis that ROS-mediated oxidative stress in atherosclerotic lesions induces various inflammatory elements, such as endothelial adhesion molecules (e.g. intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin), inflammatory cytokines and chemokines, through redox-sensitive transcription factors50). The overexpression of adhesion molecules in ECs facilitates leukocyte adhesion to ECs and monocyte chemoattractant protein-1 potentiates cellular motility in monocytes/macrophages, thereby accelerating the infiltration of these cells into lesions. Recruited macrophages incorporate low-density lipoprotein (LDL) cholesterol through micro/macro pinocytosis and phagocytosis pathways as well as scavenger receptor-mediated endocytosis51). Internalized endocytic vesicles fuse with lysosomes; accordingly, the enclosed lipoproteins are degraded through lysosomal digestion. After lysosomal degradation, acyl-coenzyme A: cholesterol acyltransferase 1 converts free cholesterol to highly hydrophobic cholesterol esters, thereby forming lipid-enriched foam cells52). In addition to the cholesterol deposition in macrophages, immune responses in immunocompetent cells including T cells, B cells and mast cells in the lesions orchestrate atherogenicity53). For instance, type 1 and type 2 T-helper cells polarize macrophages toward M1 and M2 subsets, respectively54). It was reported that M1 macrophages largely contribute to the progression of atherogenesis through its pro-inflammatory ability. Furthermore, inflammasomal production of interleukin (IL)-1β reportedly facilitates development of atherosclerotic lesions55); thus, innate immunity as well as adaptive immunity can modify the atherosclerotic vascular inflammation. We have previously reported that Capn2, Capn6 and Capn9, but not other family members, are induced in the pro-atherogenic aortae of high cholesterol diet-fed Ldlr−/− mice36). Among these calpain species, conventional calpain-2 is induced in response to oxidized or enzymatically-modified LDL or its component lysophosphatidylcholine, and is abundant in ECs in mouse and human atheromas34). The calpain inhibitors calpeptin or ALLM (N-acetyl-L-leucyl-L-leucyl-L-methioninal) appear to prevent pro-atherogenic barrier dysfunctions in ECs, and reduce the recruitment of circulating monocytes into atherosclerotic lesions; thus, the administration of these inhibitors to high cholesterol diet-fed Apoe−/− or Ldlr−/− mice suppresses the development of atherosclerotic lesions. In these cases, barrier dysfunction is caused by the calpain-2-induced proteolysis of VE-cadherin, because VE-cadherin promotes homophilic adhesion between ECs, thereby forming an interendothelial barrier56). Subramanian et al. also noted that atherosclerotic lesion progression in angiotensin II-infused Ldlr−/− mice was impaired by the calpain inhibitor BDA-41057). Transgenic overexpression of calpastatin significantly attenuated angiotensin II- or hypercholesterolemia-induced atherosclerotic development in Ldlr−/− mice58). Furthermore, myeloid calpain-1 or leukocyte calpain-2 were involved in angiotensin II-induced atherosclerosis58). In this case, calpain inhibition reduced the motility, mitosis and nuclear factor-κB (NF-κB)-dependent inflammatory responses in isolated macrophages. Collectively, it is likely that conventional calpains are involved in the pro-atherogenic regulation of EC and macrophages.
In addition to the pro-atherogenic actions of the conventional calpains, we recently identified the contribution of unconventional calpains to the pathogenesis of atherosclerosis36). Indeed, Capn6 ablation prevented the development of atherosclerotic lesions in mice, whereas Capn9 knockout did not affect the phenotype. Calpain-6 is localized to macrophages, but not to other vascular component cells in advanced human and murine atheromas. Consistently, bone marrow transplantation experiments have demonstrated that myeloid Capn6 is responsible for atherogenesis. Calpain-6 in macrophages is inducible in response to inflammatory cytokines such as tumor necrosis factor-α (TNF-α), and directly binds to CWC22, an essential loading factor for the exon-junction complex (EJC). CWC22 escorts the EJC to the nucleus, which is necessary for spliceosome-driven mRNA splicing. The efficiency of mRNA splicing was reduced after silencing CWC2259); thus, CWC22 is indispensable for mRNA splicing. The binding of calpain-6 with CWC22 in the cytoplasm prevented the nuclear translocation of CWC22. Accordingly, the splicing efficiency of their target genes, including Rac1, was decreased in the presence of calpain-6, which conferred atherogenicity to foamy macrophages. Rac1 is a negative regulator of the pinocytosis pathway; thus, the impaired nuclear translocation of CWC22 by calpain-6 augments the pinocytotic uptake of native LDL in macrophages. Importantly, the ablation of calpain-6 substantially decreased pinocytotic activity in macrophages in murine atherosclerotic lesions36). Immunohistochemistry of human aortic atheromas showed that the nuclear localization of CWC22 was diminished in macrophages in advanced atheromas but not in cells of mild atherosclerotic lesions, while calpain-6 was upregulated in macrophages in advanced atheromas36). Thus, the calpain-6-induced disruption of CWC22/EJC in macrophages translates to human atherosclerotic lesions. A previous review articles have further details of calpain-mediated regulation of atherosclerosis10) and pro-atherogenic cholesterol handling in macrophages60).
Abdominal aortic aneurysm, a representative atherosclerotic disease, is a major cause of cardiovascular death and the tenth leading cause of death in men over 65 years of age in western countries61). Excessive oxidative stress in atheroprone arteries facilitates the accumulation of matrix metalloproteinases (MMPs) and subsequent remodeling of vascular tissues, which is sometimes a threshold process in aneurysmal diseases62, 63). Indeed, macrophage- and vascular cell-derived MMPs-9 and -12 are considered major exacerbators of aneurysmal diseases64, 65), because they break down vascular structures, including the elastic lamellae and basement membrane, which leads to vascular dissection62, 63). Previous proteomic analyses demonstrated the contribution of calpain-2 to aortic aneurysms in Marfan syndrome66); thus, it was suspected that conventional calpains cause atherosclerotic aortic aneurysms. Indeed, the pharmacological intervention of conventional calpains by BDA-410 ameliorated aortic dissection in angiotensin II-infused Ldlr−/− mice, concomitant with the reduction of MMP12, IL-6 and MCP-1 levels in the vessels67). In contrast, the myeloidspecific transduction of calpastatin or deficiency of calpain-1 as well as the macrophage-specific deficiency of calpain-2 had no effect on aneurysmal formation in angiotensin II-induced Ldlr−/− mice, while deficiency of conventional calpains significantly attenuated the development of atherosclerotic lesions68). Thus, it is suspected that vascular component cells, such as ECs or VSMCs, or other immune cells are involved in calpain-dependent aortic aneurysms.
Diabetic Angiopathy
Type 1 and type 2 diabetes mellitus are chronic metabolic diseases caused by impaired insulin production in the pancreas and systemic insulin resistance, respectively, leading to the dysregulation of plasma glucose levels. It is well known that hyperglycemia results in vascular disorders, including diabetic retinopathy and nephropathy, which are the major complications of this disease5). Among the vascular component cells, dysfunction of ECs is primarily responsible for hyperglycemia-induced angiopathy69). While the exact mechanisms underlying these endothelial disorders are still unclear, the majority of studies have been based on the context that endothelial disorders are caused by excessive oxidative stress in blood vessels70). Indeed, it was reported that the exposure of ECs to high glucose conditions resulted in the elevation of ROS levels71). Furthermore, endothelial-dependent vasodilation, which is measured as an index of endothelial integrity, was reduced in diabetic animals, and was recovered by the administration of antioxidants72). Indeed, the elevation of ROS levels in blood vessels was detected in diabetic mice and human diabetic patients73, 74), suggesting a pivotal role of ROS signaling in the pathogenesis of diabetic endothelial disorders. It was previously reported that the major source of ROS in blood vessels is NADPH oxidase, xanthine oxidase and uncoupled nitric oxide (NO) synthase (NOS) as well as mitochondrial respiration75). Uncoupled eNOS, which generates superoxides instead of NO, as well as xanthine oxidase, and NADPH oxidase are largely responsible for ROS production even in diabetic vessels76, 77). It was reported that physiological NO production in ECs was dependent upon conventional calpains. Youn et al. noted that the vascular endothelial growth factor (VEGF)-induced production of NO in cultured ECs was reduced by calpain inhibitors ALLN (N-acetyl-leucyl-leucyl-norleucinal) or calpeptin78). VEGF facilitates calpain translocation to the plasma membrane and formation of the molecular complex together with ezrin; as a result, the calpain-associated molecular complex potentiates the phosphorylation of AKT, AMP-dependent kinase and endothelial NOS (eNOS)s1179, which is necessary for VEGF-induced NO production and subsequent angiogenic responses in ECs. Conversely, calpain-induced defects of endothelial NO systems have been also proposed. Cell-based experiments showed that ionophore-induced calpain overactivation proteolyzed heat shock protein 90 (HSP90) as well as eNOS and neural NOS79). Whereas the difference between the VEGF- and ionophore-induced NO production has not been thoroughly elucidated, Ca2+ overloading in cytoplasm might cause the excessive activation of conventional calpains thereby degrading unusual non-physiologic substrates including HSP90, which seems to be distinct from the physiological regulation mechanism. In addition to physiologic NO production, calpain was reportedly associated with defective NO systems in diabetes. Stalker et al. previously reported that inhibition of conventional calpains by ZLLal (aldehyde benzyloxycarbonylleucyl-leucinal) recovers impaired NO production and subsequent vasculitis in mesenteric venules in ZDF rats, concomitantly with the increasing association of eNOS with HSP9080). Chen et al. reported that conventional calpains in human umbilical vein endothelial cells were activated under high glucose conditions, accompanied by a reduction of NO production and elevation of cytosolic ROS levels without altering NOS expression levels81). Intriguingly, the glucose-induced changes in NO and ROS production were reversed by the transduction of calpastatin. Furthermore, aortic ROS levels were elevated in transgenic type 1 diabetic OVE26 mice, and were inhibited by calpastatin transduction. While endothelium-dependent vasodilation in diabetic OVE26 mice was blunted, calpastatin transduction improved the endothelial integrity of diabetic animals81). It was also documented that siRNA against calpain-1 prevented phosphorylation of eNOS at threonine 497/495 in murine aorta and cultured ECs under the hyperhomocysteinemia/high glucose conditions thereby improving NO production and subsequent EC integrity82). Thus, the overactivation of conventional calpain systems under diabetic conditions promotes dysfunctional endothelial NO systems, leading to reduction of endothelial integrity.
In addition to its defective regulation of NO signaling, the calpain-induced perturbation of prostanoid signaling has been reported as another pathogenic cue for diabetic angiopathy. Randriamboavonjy et al. reported that pharmacological calpain inhibition by A-705232 recovered EC-dependent vasodilation in diabetic mesenteric arteries83). This recovery was dependent upon protection by prostaglandin I2 synthase (PGIS) from calpain-induced degradation. While the treatment of mesenteric arteries with peroxynitrate donors impaired endothelium-dependent vasodilation even in the presence or absence of a NO scavenger83), it was recovered by the calpain inhibitor calpeptin. Collectively, the overactivation of conventional calpains opposes prostaglandin I2 synthesis in diabetic small vessels independent of NO signaling, which might cause diabetic angiopathy.
Proliferative Retinopathy and Cancer Neovessels
It is well known that abnormalities of pathological neovessels in proliferative retinopathy and tumor neovessels are caused by defective angiogenic signaling in ECs. During normal physiological status, ECs rearrange their shape via oxygen sensors and hypoxia-inducible factors, including prolyl hydroxylase domain 2 and hypoxia-inducible factor (HIF)-2α, respectively to optimize blood flow84). When the tissue becomes hypoxic, angiogenic responses are robustly activated through angiogenic mediators including VEGF-A, VEGF-C, fibroblast growth factors and angiopoietin-2. Such mediators accelerate the motility and mitosis of leading ECs (termed the tip cell) mainly through their surface receptors thereby driving endothelial tube formation. Tip cells guide the following ECs (termed stalk cells) and VEGFR2 in stalk cells is subsequently downregulated through the DLL4/NOTCH pathway to facilitate the coverage of immature vessels with pericytes84). According to such stepwise vascular maturation, the physiological neovessels acquire a hierarchical network as shown in the pre-existing vasculature. Increasing evidence suggests that conventional calpain systems contribute to physiological angiogenesis. For example, Zheng et al. recently noted the calpain-induced acceleration of HIF-1 signals85). In this case, calpain-induced the proteolysis of filamin-A, a large cytoskeletal actin-binding protein, whereby filamin-A proteolytic fragments physically interacted with HIF-1α to promote the nuclear localization of HIF-1α. However, it is noteworthy that Capn1-deficient mice as well as EC-specific Capn2-deficient mice did not exhibit the lethal vascular defects23, 26); thus, the impact of calpain-dependent angiogenesis on physiological vascular regulations is currently unclear. Please refer to an excellent review for further details of calpain-mediated physiological angiogenesis86).
In contrast to the physiological neovessels, pathological neovessels, which are frequently detected in inflamed lesions (e.g. atherosclerotic lesions and diabetic retinopathy) as well as tumor tissues, exhibit an unstable and non-hierarchical structure84, 87, 88). Although both physiological and pathological angiogenesis are similarly driven by common angiogenic cues (e.g. angiopoietin-2 and VEGF), pathological neovessels exhibit immature properties characterized by a lack of pericyte coverage, a thin extracellular matrix layer and poor inter-endothelial junctions84). Because of these structural instabilities, pathological neovessels provide a major route for the recruitment of immune cells into inflamed lesions or for metastatic cancer cell emigration from primary tumor tissues, which leads to the further progression of disease. The critical difference between physiological and pathological angiogenesis is that the latter is affected by the surrounding inflammatory conditions. Interestingly, inflammatory and angiogenic signals are slightly interrelated and redundant. Indeed, several classes of proinflammatory cytokines, including TNF-α and IL-6, have angiogenic effects on vascular ECs89–91). Furthermore, the ablation of endothelial SOCS3, an endogenous inhibitor of JAK/STAT cytokine signals, accelerated growth factor- and cytokine-induced angiogenesis thereby aggravating cancer growth and oxygen-induced retinopathy (OIR) in mice90). It was reported that conventional calpains are associated with pathological angiogenesis. Indeed, Hoang et al. noted that calpain inhibitors MDL 28170, PD150606, or ALLN opposed the pathogenesis of OIR in mice, and normalized the morphology of retinal vessels92). Furthermore, we previously documented the contribution of endothelial calpains to pathological angiogenesis35). Mechanistically, the loss of calpastatin by several growth factor classes, causes the calpain-1-induced proteolytic degradation of SOCS3, leading to VEGF-C production through excessive JAK/STAT signaling. Accordingly, calpastatin downregulation facilitates IL-6-driven angiogenic responses in ECs through the local autocrine action of VEGF-C. Similarly, the downregulation of calpastatin and SOCS3 expression is detectable in neovessels in human colon adenocarcinoma, lung adenocarcinoma and malignant astrocytoma. The EC-specific transduction of calpastatin counteracts the STAT3/VEGF-C axis and antagonizes pathological angiogenesis in allograft tumors and OIR in mice, which suppresses such diseases. Whereas pathological neovessels in these diseases lack coverage of pericytes and formation of a basement membrane, calpastatin transduction improves the maturity of neovessels. Therefore, the overactivation of conventional calpains sensitizes JAK/STAT inflammatory cascades, which converts angiogenic ECs to pathological status.
In addition to angiogenic roles in ECs, conventional calpains are associated with the metabolism of angiostatic mediators. Saito et al. reported that calpain in cancer cells proteolytically degraded the angiostatic peptide vasohibin-133). Vasohibin-1 synthesized in ECs can inhibit angiogenic responses by an autocrine mechanism; thus, the degradation of vashibin-1 by tumoral calpains might facilitate tumor angiogenesis.
Vascular Fibrosis and Pulmonary Hypertension
It is well known that aging induces structural and functional changes in arteries, and is a critical risk factor for cardiovascular defects, such as hypertension and atherosclerosis93, 94). An age-associated increase in arterial wall stiffness is largely dependent upon remodeling of the ECM and vascular calcification. In general, ECM remodeling in aged arteries occurs because of increased collagen content, elastin fragmentation, and is triggered by the transformation of contractile VSMCs to a synthetic phenotype. Letavernier et al. reported that vascular fibrosis and hypertrophy in angiotensin II-infused mice was prevented by calpastatin transduction95). Furthermore, impaired fibrogenic responses were caused by a reduction of medial MMP levels and NF-κB activity. Similarly, Jiang et al. reported that the induction of calpain-1 in VSMCs was associated with MMP2 upregulation and subsequent ageassociated vascular fibrosis in rats19). Furthermore, the overexpression of calpain-1 in rats accelerated arterial calcification as well as fibrosis in aged rats, concomitant with a reduction of osteopontin and osteonectin levels in VSMCs96). Tang et al. reported that the transduction of calpastatin reduced VSMC proliferation and collagen synthesis thereby inhibiting restenosis induced by carotid artery ligation in mice97). Thus, calpain systems contribute to the variety of fibrogenic and proliferative responses in VSMCs.
Pulmonary hypertension is a proliferative vascular disease characterized by medial thickening and severe fibrosis in the right ventricular artery thereby increasing pulmonary vascular resistance and subsequent right heart failure98, 99). Excessive proliferation of VSMCs and hypertrophy as well as the production of ECM contributes to medial hypertrophy, leading to the destruction of precapillary pulmonary arteries and a sustained elevation of pulmonary arterial pressure. Ma et al. previously reported that a deficiency of Capns1 reduced both calpain-1 and -2 levels, reduced collagen synthesis and remodeling in pulmonary arterioles and the subsequent pathogenesis of pulmonary hypertension in mice100). Calpain activated transforming growth factor (TGF)-β1 via direct proteolytic processing; thus, calpain deficiency resulted in the prevention of TGF-β1-induced fibrogenic responses in VSMCs. They also reported that calpain-2 activated Akt via the TGF-β1-mTORC2 pathway in pulmonary artery smooth muscle cells101). However, patients suffering from idiopathic pulmonary arterial hypertension exhibited high plasma calpastatin levels102). The hepatocyte-specific transduction of calpastatin reduced pulmonary calpain activity, resulting in the amelioration of murine hypoxia-induced pulmonary hypertension102). Thus, reduced extracellular calpastatin levels as well as calpain activity in pulmonary arterioles may contribute to the pathogenesis of pulmonary hypertension.
Conclusive Remarks
In conclusion, the dysregulation of calpain systems is responsible for a variety of degenerative vascular disorders (Fig. 2), while these calpain systems are not substantially associated with vascular regulation under physiological conditions as well as vascular development. It is likely that the excessive activity of conventional calpains can induce these disorders, which are commonly caused by exogenous stressors, such as inflammatory cytokines and growth factors. Furthermore, loss of the endogenous inhibitor calpastatin potentiates calpain activity in some instances. In particular, it is noteworthy that the calpain-induced dysfunctional integrity of ECs and fibrogenic responses in VSMCs were reproduced by multiple studies (Table 1). In contrast to conventional isozymes, calpain-6 contributes to atherogenesis through its interference of mRNA splicing in macrophages. This indicates that calpain-6 might be associated with other macrophage-related diseases. Importantly, the loss-of-function of calpains almost uniformly ameliorates degenerative disorders. Indeed, the transgenic overexpression of calpastatin as well as calpain inhibitors such as MDL28170 and BDA-410, are effective treatments for degenerative disorders, suggesting that calpain inhibitors are potentially promising for the treatment of chronic vascular diseases. Candidate compounds for inhibiting conventional calpains are currently being tested in clinical trials including those of neurodegenerative diseases14). These agents should be repositioned for cardiovascular fields in the future, while agents that inhibit calpain-6 are currently unavailable. The development of subtype selective inhibitors, in particular those targeting unconventional calpains, is indispensable for treating atherosclerosis and related diseases.
Fig. 2.
Overview of the regulation of degenerative vascular disorders by calpain systems. LDL: low-density lipoprotein, EC: vascular endothelial cells, VSMCs: vascular smooth muscle cells, NFκB: nuclear factor κB, IκB: inhibitor κB, SOCS3: suppressor of cytokine signalling 3, JAK/STAT: Janus kinase/signal transducer and activator of transcription, NOS: nitric oxide synthase.
Table 1. Possible contribution of calpain systems in degenerative vascular disorders.
Disease type | Experimental strategy | Intervention | Target molecule | Outcomes | Ref. |
---|---|---|---|---|---|
Atherosclerosis | Apoe−/− mice+high fat diet | Calpeptin | VE-cadherin | Atherosclerotic lesion↓ | 34 |
Ldlr−/− mice+high fat diet | ALLM | Aortic macrophage recruitment↓ | |||
Endothelial permeability↓ | |||||
Human umbilical vein endothelial cells+lysophosphatidylcholine | Capn2 siRNA | VE-cadherin | Endothelial permeability↓ | 34 | |
VE-cadherin proteolysis↓ | |||||
Ldlr−/− mice+high fat diet | Capn6 −/− | CWC22 | Atherosclerotic lesion↓ | 36 | |
Aortic macrophage recruitment↓ | |||||
Pinocytosis activity↓ | |||||
Bone marrow-derived macrophages +TNF-α | Capn6−/− | CWC22 | Cellular motility↑ | 36 | |
Pinocytosis activity↓ | |||||
Rac1 activity↑ | |||||
Nuclear localization of CWC22↑ | |||||
Ldlr−/− mice+high fat diet+AngII | BDA-410 | – | Atherosclerotic lesion↓ | 57 | |
Ldlr−/− mice+high fat diet+AngII | CAST+/0 (myeloid specific) | – | Atherosclerotic lesion↓ | 58 | |
Capn1−/− (myeloid specific) | |||||
Capn2flox/flox/LysM-Cre+/0 | |||||
Ldlr−/− mice+high fat diet | CAST+/0 | – | Atherosclerotic lesion↓ | 58 | |
Abdominal aortic aneurysm | Ldlr−/− mice+high fat diet+AngII | BDA-410 | – | Abdominal aneurysm↓ | 57 |
Aortic macrophage recruitment↓ | |||||
MMP12↓ | |||||
Proinflammatory genes↓ | |||||
Ldlr−/− mice+high fat diet+AngII | CAST+/0 (myeloid specific) | – | Abdominal aneurysm→ | 58 | |
Capn1−/− (myeloid specific) | Aortic macrophage recruitment↓ | ||||
Capn2flox/flox/LysMCre+/0 | |||||
Bone marrow-derived macrophages | CAST+/0 | IκBα | NF-κB cascade↓ | 58 | |
Diabetic angiopathy | Zucker diabetic fatty rats (type 2 diabetes) | ZLLal | eNOS | NO production↑ | 80 |
EC-leukocyte interaction↓ | |||||
Human umbilical vein endothelial cells+high glucose | Cast transduction | – | Nitric oxide production↓ | 81 | |
MDL28170 | ROS production↓ | ||||
OVE26 type 1 diabetic mice | CAST+/0 | – | Aortic ROS production↓ | 81 | |
EC-dependent vasodilation↑ | |||||
Hyperhomocysteinemic/diabetic mice (cystathionine β-synthasedeficient mice+high-methionine diet+streptozotocin) | MDL28170 | – | EC-dependent vasodilation↑ | 82 | |
Calpeptin | ROS production↓ | ||||
ALLM | |||||
Human aortic endothelial cells | MDL28170 | – | NO production↑ | 82 | |
CAPN1 siRNA | P-eNOS-pThr495↓ | ||||
Streptozocin-induced diabetic mice | A-705232 | PGI2 synthase | EC-dependent vasodilation↑ | 83 | |
Diabetic New Zealand obese mice | Calpeptin | PGI2 synthesis↑ | |||
Pathological angiogenesis | Oxygen-induced retinopathy model in mice | MDL 28170 | – | Oxygen-induced retinopathy↓ | 92 |
PD150606 | Retinal vascular permeability↓ | ||||
ALLN | |||||
Oxygen-induced retinopathy model in mice | LNL-CAST+/0/Tie2-Cre+/0 | SOCS3 | Tumor angiogenesis↓ | 35 | |
Oxygen-induced retinopathy↓ | |||||
Tumor implantation in mice | VEGF-C production↓ | ||||
Maturation of neovessels↑ | |||||
Human aortic endothelial cells+ IL-6 | Cast siRNA | SOCS3 | Migration↑ | 35 | |
Proliferation↑ | |||||
Tube formation↑ | |||||
JAK/STAT3 signal↑ | |||||
VEGF-C production↑ | |||||
Co-culture B16 melanoma cells with ECs | MDL28170 | Vasohibin-1 | Vasohibin-1 proteolysis↓ | 33 | |
Age-associated fibrosis | AngII-induced aortic hypertrophy model | CAST+/0 | – | Aortic fibrosis↓ | 95 |
Aortic hypertrophy↓ | |||||
Perivascular leukocyte recruitment↓ | |||||
NF-κB cascade↓ | |||||
Primary VSMCs from aged rats+ AngII | Calpain inhibitor I | – | MMP2 activity↓ | 19 | |
CAST transduction | VSMC migration↓ | ||||
Primary VSMCs from aged rats | CAPN1 transduction | – | Fibrosis↑ | 96 | |
Cultured carotid artery rings | Calcification↑ | ||||
Smad1/2↑ | |||||
TGF-β1↑ | |||||
Osteopontin↓ | |||||
Osteonectin↓ | |||||
Arterial restenosis | Carotid artery ligation model in mice | CAST+/0 | – | Arterial restenosis↓ | 97 |
Arterial fibrosis↓ | |||||
Arterial hypertrophy↓ | |||||
Arterial MMP2/TGF-β1↓ | |||||
Primary VSMCs from mice+ PDGF-BB | Capn1 siRNA | – | Proliferation↓ | 97 | |
Capn2 siRNA | Migration↓ | ||||
Pulmonary hypertension | Chronic hypoxia-induced pulmonary hypertension model in mice | Capn2flox/flox/ER-Cre+/0 | TGF-β1 | Pulmonary hypertension↓ | 100 |
Collagen I in pulmonary arterioles↓ | |||||
Hypertrophy in pulmonary arterioles↓ | |||||
Monocrotaline-induced pulmonary hypertension model in rats | MDL28170 | – | Pulmonary hypertension↓ | 100 | |
Hypertrophy in pulmonary arterioles↓ | |||||
Human pulmonary arterial smooth muscle cells+PDGF-BB | MDL28170 | TGF-β1 | Collagen I synthesis↓ | 100 | |
Capn1 siRNA | Proliferation↓ | ||||
Capn2 siRNA | TGF-β1 production↓ | ||||
Chronic hypoxia-induced pulmonary hypertension model in mice | Capn2flox/flox/ER-Cre+/0 | – | P-Akt-S473 in pulmonary arterioles↓ | 101 | |
P-Akt-T308 in pulmonary arterioles↓ | |||||
Human pulmonary arterial smooth muscle cells+PDGF-BB | MDL28170 | – | Collagen I synthesis↓ | 101 | |
Capn2 siRNA | Proliferation↓ | ||||
P-Akt-S473↓ | |||||
P-Akt-T308↓ | |||||
Murine hypoxia-induced pulmonary hypertension model | CAST transduction (CMV promoter) | – | Pulmonary hypertension↓ | 102 | |
Hypertrophy in pulmonary arterioles↓ | |||||
SM22-5HTT1 mice | CAST transduction (CRP promoter) | ||||
PD150606 | |||||
Murine pulmonary arterial smooth muscle cells+FBS, +PDGF-BB, +EGF | PD150606 | – | Proliferation↓ | 102 |
Acknowledgements
This study was supported in part by JSPS KAKENHI Grant Number 26461368 (to A.M.), JSPS KAKENHI Grant Number 15K09418 (to T.M.), a research grant from Takeda Science Foundation, a research grant from Banyu Life Science Foundation International, the Japan Heart Foundation & Astellas Grant for Research on Atherosclerosis Update, and SENSHIN Medical Research Foundation (all to T.M.).
Conflict of Interest
None.
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