Current Angiogenesis, 2012, 1, 39-45 39 Regulation of Angiogenesis by the Small B-

Lothar C. Dieterich and Anna Dimberg*

1Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden

Abstract: B-crystallin is a member of the small heat shock protein family, primarily known for their ability to bind to and stabilize un- or misfolded proteins and protect cells from protein denaturation-induced damage. Besides its general function, B-crystallin has been assigned additional activities, such as inhibition of apoptosis, cytoskeletal sta- bilization and turnover, and ubiquitination and degradation of specific target proteins. B-crystallin is frequently ex- pressed in endothelial cells and perivascular cells, and can be further induced by pro-angiogenic growth factors and differ- ent types of cellular stress. Recently, B-crystallin has been implicated in the regulation of angiogenesis and blood vessel function by endothelial cell intrinsic and extrinsic mechanisms. The aim of this review is to summarize some of the recent findings concerning the various functions of B-crystallin, with a specific focus on endothelial cell biology and regulation of angiogenesis. Keywords: B-crystallin, angiogenesis, apoptosis, chaperone, endothelial, heat shock protein, stress, VEGF.

INTRODUCTION Table 1. -crystallin Type Small Heat Shock Proteins Ex- B-crystallin is a small heat shock protein that affects a pressed in Human plethora of cellular processes by acting as a molecular chap- erone, controlling the stability and activity of other proteins. Systematic Name Alternative Name B-crystallin is constitutively expressed in several tissues and cell types. In addition, it is up-regulated by various kinds HSPB1 of cellular stress such as heat shock, osmotic stress, and oxi- dative stress, and is generally believed to protect cells from HSPB2 Myotonic dystrophy protein kinase binding stress-induced damage. Thus, B-crystallin plays an impor- protein (MKBP) tant role in various pathological conditions, including ische- mia, chronic inflammation of the brain, and cancer. In addi- HSPB3 HspL27 tion, B-crystallin has recently emerged as a regulator of HSPB4 A-crystallin angiogenesis, through both endothelial cell intrinsic and ex- trinsic mechanisms. This formerly unappreciated role of B- HSPB5 B-crystallin crystallin is the focus of this review. HSPB6 Hsp20

THE -CRYSTALLIN TYPE SMALL HEAT SHOCK HSPB7 cardiovascular Hsp (cvHSP) PROTEIN FAMILY HSPB8 Hsp22, H11

B-crystallin is a member of the highly conserved - HSPB9 Cancer testis antigen 51 crystallin type heat shock protein (-HSPs) family. Members of this protein family can be found in almost all life forms, HSPB10 Outer dense fiber protein 1 (ODF) from bacteria and archaea up to plants and animals [1]. So HSPB11 Placental protein 25 (PP25) far, eleven -HSPs have been identified in humans (Table 1) [2, 3]. Proteins of this family are often merely referred to as List of systematic and alternative names of the 11 -crystallin type small heat shock “small heat shock proteins” due to their low molecular proteins that have been identified in human so far. weight (typically ranging between 12 and 40 kDa). This however is not completely accurate as there are some low constitutively expressed in many other tissues, including molecular weight heat shock proteins that bear no structural skeletal and cardiac muscle, brain, and kidney. Furthermore, resemblance to the -HSPs, for example bacterial Hsp33 and -HSP expression is inducible in many cell types in response Hsp15. The name "crystallin" reflects the original finding to stress or other extracellular stimuli. B-crystallin may that some members of the family are particularly highly ex- also be expressed in endothelial cells, suggesting a role in pressed in the vertebrate eye lens. However, -HSPs are also regulation of angiogenesis and vascular function [4, 5].

*Address correspondence to this author at the Department of Immunology, STRUCTURE OF B-CRYSTALLIN Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden; Tel: +46 18 471 4002; Fax: +46 18 55 89 31; B-crystallin is a true heat shock protein and one of the E-mail: [email protected] most studied members of the -HSP family. The C-terminal

2211-5536/12 $58.00+.00 © 2012 Bentham Science Publishers 40 Current Angiogenesis, 2012, Vol. 1, No. 1 Dieterich and Dimberg half of the protein contains the name-giving highly con- tain normal function of tissues, particularly the eye lens and served -crystallin domain that is shared by all -HSP fam- muscle, is illustrated by the drastic consequences of a mis- ily members (Fig. 1). The -crystallin domain forms - sense mutation in the -crystallin domain, R120G. This mu- sandwich structures and mediates tight homophilic interac- tation alters the oligomerization of B-crystallin and disturbs tions which allow the formation of homo- and hetero-dimeric its chaperone function, and has been linked to cataracts as units. These dimers in turn serve as building blocks for well as skeletal and cardio-myopathy, characterized by for- higher order oligomers [6]. In contrast to the C-terminus, the mation of inclusion bodies rich in aggregated B-crystallin structure of the N-terminal domain is less defined and does and other proteins [20-24]. Furthermore, B-crystallin has not form multimers in isolation, but may contribute to the been shown to affect multiple cellular functions through structural heterogeneity observed in B-crystallin complexes binding to key proteins regulating apoptosis, the cytoskele- [7]. It contains a WPDF domain, named after an amino acid ton and proteasomal degradation, and by modulating their motif consisting of tryptophane, aspartate, proline and phen- activity. ylalanine, which is moderately conserved among the -HSPs [8]. Inhibition of Apoptosis by B-crystallin B-crystallin contains three conserved serine phosphory- B-crystallin has been shown to inhibit apoptosis in sev- lation sites (Ser19, Ser45 and Ser59), each being phosphory- eral ways. For example, B-crystallin binds to pro-apoptotic lated downstream of distinct signaling pathways, for exam- Bcl2 family members and prevents their translocation to the ple in response to toxic chemicals, heat shock, or growth mitochondrial membrane [25]. Furthermore, B-crystallin factor stimulation. Differential serine phosphorylation regu- has been demonstrated to directly bind to procaspase-3 and lates the chaperone function of B-crystallin and modulates to prevent its auto-catalytic cleavage and activation, thereby its ability to bind to and affect the function of other proteins, inhibiting both extrinsic and intrinsic apoptotic pathways which will be briefly discussed in the following sections. For [26-28]. Notably, the Ser59 phosphorylation site, which is a more extensive review of the topic, see Gusev et al., [8] regulated downstream of p38/MAPKAP-kinase 2 activation, and Arrigo et al. [9]. is necessary and sufficient for inhibition of caspase-3 activa- tion and the pro-survival function of B-crystallin [29-32]. B-CRYSTALLIN CHAPERONE ACTIVITY AND In addition, B-crystallin increases cell survival by inhibit- FUNCTION ing oxidative stress, at least partially through raising the in- tracellular level of glutathione [33, 34]. -HSPs primarily act as molecular chaperones, protect- ing cells from stress induced protein denaturation, aggrega- tion and subsequent damage [1, 10, 11]. -HSPs lack direct Regulation of the Cytoskeleton and Muscle Function refolding capacity. Their main task is to bind to and stabilize B-crystallin binds to and stabilizes various components partially unfolded proteins via their flexible N-terminal do- of the cytoskeleton, including actin, vimentin, and microtu- main and the -crystallin domain until they become refolded buli, and modulates their assembly and dynamics [16, 35, by ATP-dependent large HSPs (such as ) or are di- 36]. These interactions are not only important for protecting rected to the proteasome for degradation [12]. B-crystallin cells from stress induced damages of the cytoskeleton but chaperone activity has been shown to be dependent on the has also been implicated in the regulation of cytoskeletal oligomerization state of the protein that in turn is dependent turnover, cellular locomotion, and function of muscle cells on serine phosphorylation [13-19]. Its importance to main- [8].

Figure 1: B-crystallin protein structure and phosphorylation sites. Schematic overview of the protein structure of B-crystallin, indi- cating the position of the WPDF and the -crystallin domain. The three Serine phosphorylation sites (Ser19, Ser45, Ser59) are represented by black dots together with the major kinases responsible for phosphorylation of those residues. B-crystallin in Angiogenesis Current Angiogenesis, 2012 Vol. 1, No. 1 41

B-crystallin in Protein Ubiquitination and Degradation B-CRYSTALLIN EXPRESSION IN ENDOTHELIAL AND PERIVASCULAR CELLS B-crystallin facilitates ubiquitination and proteasomal degradation of specific proteins, which potentially may af- B-crystallin is expressed in primary endothelial cells in fect numerous cellular functions and signaling pathways. culture, particularly in microvascular endothelial cells from B-crystallin has been found to accumulate in cytoplasmic various species such as human dermal microvascular endo- "aggregosomes", a characteristic feature of some neurode- thelial cells, bovine capillary endothelial cells, and murine generative diseases and muscular dystrophies, which also myocardial endothelial cells. In contrast, venous endothelial contain many ubiquitinated and misfolded proteins [37]. In- cells such as human umbilical vein endothelial cells and hu- teractions between B-crystallin and ubiquinated proteins man saphenous vein endothelial cells express low levels of probably reflect the chaperone function of -HSPs men- B-crystallin, if any (unpublished observation). Expression tioned above, namely to bind and stabilize proteins before can be induced by various types of stress including osmotic their proteasomal degradation. Additionally, B-crystallin is stress, arsenite treatment and dicarbonyl stress and has been directly involved in the process of protein ubiquitination and shown to protect endothelial cells from glucose-induced degradation via interaction with the F-box protein 4 (FBX4), apoptosis [4, 34, 52]. Similarily, increased vascular expres- a component of an SKP1/-1/F-box (SCF) sion of B-crystallin is induced in various pathological con- ligase. This interaction is increased by phosphorylation on ditions where endothelial cells and pericytes are exposed to Ser19 and Ser45 [38, 39]. The responsible kinases that medi- stress. For example, while being lowly expressed in the nor- ate phosphorylation at these Ser residues have not been fully mal brain vasculature, B-crystallin is induced in endothelial characterized, but in the case of Ser45 have been shown to cells surrounding gliotic tissue of cerebral arteriovenous include p42/44 and PKC- [29, 40]. Furthermore, phos- malformations [53]. In cerebral autosomal dominant arte- phorylation of Ser45 is modulated during cell cycle progres- riopathy with subcortical infarcts and leukoencephalopathy sion [29]. Interestingly, the SCF-B-crystallin complex has (CADASIL), B-crystallin is expressed in vascular myo- been reported to catalyze the ubiquitination of cell cycle cytes of the affected arteries [54]. regulatory proteins, for example cyclin D1, and to target In addition to stress, the pro-angiogenic growth factors them for proteasomal degradation [41]. Thus, B-crystallin FGF-2 and VEGF induce robust up-regulation of B- is likely involved in regulation of cell cycle progression, but crystallin in microvascular endothelial cells [5]. In accor- might also affect the degradation of other, hitherto unidenti- dance with this, B-crystallin is up-regulated in endothelial fied proteins. cells in human tumors, including clear cell lung cancer, renal cancer and multiple myeloma [5, 55] and in breast cancer TRANSCRIPTIONAL REGULATION OF B- xenografts in mice [49]. CRYSTALLIN EXPRESSION B-crystallin is highly expressed in the eye lens, where ENDOTHELIAL CELL INTRINSIC AND EXTRINSIC tissue-specific expression is regulated by Pax6, Maf and FUNCTIONS OF B-CRYSTALLIN DURING ANGI- RAR/RXR complexes via lens-specific regulatory regions OGENESIS [42, 43]. However, B-crystallin is transcriptionally up-  Through its multiple activities as a molecular chaperone, regulated in many different cell types in response to cellular B-crystallin modulates angiogenesis by several different stress and factors in the cellular microenvironment. Expres- mechanisms, acting both directly in endothelial cells and sion of the cryab gene is regulated in response to heat shock, indirectly by affecting other cell types (Fig. 2). proteotoxicity and different kinds of cellular stress through the transcription factors Heat Shock Factor (HSF1), Nuclear factor of activated T-cells (NFAT) and Lens epithelium- Endothelial Cell Intrinsic Function of B-crystallin in Angiogenesis derived growth factor (LEDGF) [44-46]. Interestingly, B- crystallin has also been shown to be a p53 target gene, which The well-established role of B-crystallin in inhibiting might at least partially explain its up-regulation in several apoptosis of endothelial cells appears to be important during types of human tumors, and it appears to be required for p53- angiogenesis. B-crystallin is required for efficient tubular dependent apoptosis [47]. Signaling pathways activating morphogenesis of endothelial cells in response to pro- p38, including those activated by the pro-angiogenic factors angiogenic growth factors and paracrine stimulation from vascular endothelial growth factor (VEGF) and basic fibro- human breast cancer cells in vitro [5, 49]. Decreased ability blast growth factor (FGF-2), induce B-crystallin expres- to form tubular structures in a 3D-collagen matrix in re- sion, likely through activation of ATF6 binding to stress re- sponse to VEGF in absence of B-crystallin is associated sponse elements in the promoter of the gene [5, 30, 48, 49]. with increased endothelial apoptosis [5]. Similarly, tumors Regulation in response to pro-inflammatory signaling ap- formed in B-crystallin-deficient mice are poorly vascular- pears to be more complex and might at least in part be cell ized and many vessels are only partially lined by endothelial type dependent. For example, while the pro-inflammatory cells. Staining with an antibody recognizing cleaved caspase- cytokines interleukin 1- and tumor necrosis factor (TNF)- 3 demonstrated that endothelial cells are frequently apoptotic down-regulate B-crystallin expression in chondrocytes in tumors grown in B-crystallin-deficient mice. In addition [50], expression is induced by TNF- in astrocytes [51]. to its pro-survival function during angiogenesis, it is likely that B-crystallin affects the extensive cytoskeletal re- arrangements leading to formation of the vascular tube.

42 Current Angiogenesis, 2012, Vol. 1, No. 1 Dieterich and Dimberg

Figure 2: Schematic overview of the major functions of B-crystallin during angiogenesis. Endothelial cell intrinsic functions (inhibi- tion of apoptosis, cytoskeletal regulation) are depicted on the right, endothelial cell extrinsic functions (regulation of inflammation, platelet activation, and perivascular cells) on the left. Stabilization of pro-angiogenic growth factors may occur both in endothelial cells as well as surrounding cells and is thus depicted in the middle of the scheme.

B-crystallin co-localizes with vimentin in endothelial cells FGF-2 [58]. The in vivo relevance of this finding has been during tubular morphogenesis, but the functional relevance supported recently by Kase et al., who analyzed the retinal of this finding has not yet been investigated [5]. vasculature in B-crystallin-deficient mice [59]. In this re- port, retinal vascular density was slightly decreased in the B-crystallin in Pericytes and Vascular Smooth Muscle absence of B-crystallin, which was associated with de- Cells creased secretion of VEGF from retinal pigment epithelial (RPE) cells. VEGF and B-crystallin were co-immuno- In agreement with its high expression in muscle tissue, precipitated from cultured RPE cells, and VEGF stability B-crystallin is frequently expressed in pericytes and vascu- was decreased in the absence of B-crystallin. Very recently, lar smooth muscle cells (VSMC) [56]. Pericytes are recruited B-crystallin was shown to maintain intracrine VEGF stimu- to newly formed vessels by platelet derived growth factor lation of endothelial cells, thus conferring anti-VEGF resis- (PDGF-) secretion from endothelial cells binding to PDGFR- tance in breast cancer [49]. Knock-down of B-crystallin in B expressed on pericytes. Pericytes in turn stimulate endo- human microvascular endothelial cells in vitro was associ- thelial cells with Angiopoietin 1, contributing to vascular ated with decreased production of VEGF in response to stability, and may also be involved in guidance of the endo- paracrine stimulation from breast cancer cells. Intracrine thelial sprout [57]. The role of B-crystallin in pericytes and VEGF-stimulation of endothelial cells has an important pro- VSMCs has not been investigated, but it is likely that it af- survival function, evidenced by systemic vascular patholo- fects VSMC contractility and/or survival through its chaper- gies in mice harboring an endothelial-specific knockout of one function. Decreased pericyte/VSMC function or viability VEGF (VEGFECKO) [60]. Interestingly, aging vasculature in likely contributes to the detrimental effects on tumor angio- VEGFECKO mice show signs of endothelial apoptosis remi- genesis noted in B-crystallin-deficient mice. niscent of the tumor vasculature in B-crystallin-deficient mice. However, endothelial-specific knockout of VEGF re- B-crystallin in Regulation of Pro-Angiogenic Growth sults in severe vascular defects, while B-crystallin defi- Factor Stability ciency has not been associated with substantial vascular de- Several reports suggest that B-crystallin may play an fects under physiologic conditions, indicating that B- important role in angiogenesis by acting as a chaperone for crystallin is not strictly required for intracrine VEGF- VEGF and other growth factors. Initially, by using a protein- signaling. In the study by Kase et al., it has been suggested pin array, Ghosh et al. showed that B-crystallin derived that B-crystallin binds to unfolded VEGF and re-directs it peptides interact with the pro-angiogenic factors VEGF and to the endoplasmatic reticulum (ER), where it is refolded and B-crystallin in Angiogenesis Current Angiogenesis, 2012 Vol. 1, No. 1 43 further processed for secretion [59, 61]. However, it is not reported in various types of cancer, including glioblastoma, entirely clear in which cellular compartment B-crystallin squamous carcinoma of the head and neck, breast cancer, would bind to and stabilize VEGF in vivo, as intracellular ovarian cancer, osteosarcoma and leukemia [72-76]. Due to localization of B-crystallin and misfolded VEGF (e.g. in its strong cytoprotective functions, B-crystallin is likely to the ER or in the cytoplasm) have not been unequivocally protect tumor cells from apoptosis, limiting efficiency of shown. Further investigations are needed to clarify this ques- chemotherapy and anti-tumor immune responses and thereby tion. promoting tumor growth. Accumulating experimental and clinical data demonstrate that this is indeed the case [9, 10]. Regulation of Platelet Activation and Inflammation Accordingly, in several tumor types, B-crystallin expres- sion is associated with a poor prognosis [77-79]. Ectopic Another way through which B-crystallin may affect expression of B-crystallin in immortalized mammary angiogenesis is by interfering with platelet activation. In epithelial cells induced their transformation, suggesting that addition to its intracellular functions, B-crystallin has re- B-crystallin may act as an oncoprotein [78]. Therefore, B- peatedly been assigned extracellular functions. One of the crystallin has been suggested as target for the development earliest reports of B-crystallin being present in plasma de- of future anti-cancer drugs [9]. The recent evidence demon- scribes B-crystallin being released from injured vessels and strating that B-crystallin is induced in tumor blood vessels acting as a regulator of platelet function through reduction of and regulates endothelial survival and angiogenesis suggests platelet aggregation induced by thrombin or botrecetin [62]. that pharmacological targeting of B-crystallin may have a Subsequently, extracellular B-crystallin was shown to sup- dual advantage, by inhibiting survival and growth of tumor press ADP-induced granule secretion from human platelets cells and attenuating tumor angiogenesis. However, B- [63]. Platelets contain both pro-angiogenic and anti- crystallin is expressed in several cellular compartments, and angiogenic molecules, and it has been shown that secretion it is therefore difficult to predict how inhibition of aB- of these may be regulated during wound healing and in tu- cystallin may affect the tumor microenvironment in different mors [64]. types of cancer. Importantly, the possible impact of B- The close connection between inflammation and angio- crystallin on inflammatory processes in tumors remains to be genesis imply that proteins that alter the inflammatory re- determined. sponse may also affect angiogenesis. Accumulating data from studies on multiple sclerosis (MS) and experimental FUTURE DIRECTIONS autoimmune encephalitis (EAE), a mouse model of MS, in- B-crystallin is the first member of the small heat shock dicate that B-crystallin modulates inflammatory responses, protein family to be implicated in the regulation of angio- at least in the brain [65]. B-crystallin is constitutively ex- genesis in vivo, and several reports have shown an indisput- pressed at low levels in the central nervous system, but is able role of B-crystallin in regulating endothelial survival. strongly up-regulated during MS, and can be found in cere-  However, it is likely that B-crystallin, through its versatile brospinal fluid of MS patients and mice with EAE [66-69].  function as a molecular chaperone, has additional effects on A recent paper demonstrated increased inflammation and a the phenotype of endothelial cells, e.g. in regulation of cy- worse course of disease during EAE in B-crystallin- deficient mice, associated with increased activation of nu- toskeletal rearrangement, protein ubiquitination and growth factor signaling. Distinct expression patterns of B-crystallin clear factor  B (NF-B) signaling in the absence of B- in tumor blood vessels may influence endothelial function in crystallin [68]. Further support for an anti-inflammatory tumors. Therefore, it is important to understand what conse- function of B-crystallin has recently been presented by quences this may have on the tumor microenvironment. Arac and colleagues who showed that administration of re- combinant B-crystallin 12 h after experimental induction of According to several studies, B-crystallin has an ex- stroke in mice reduced stroke volume and inflammatory cy- tracellular function. The potential role of extracellular B- tokines [70]. Consequently, B-crystallin-deficient mice crystallin in angiogenesis has not yet been addressed. B- suffered from increased lesion size and more severe stroke crystallin has been found in serum from patients with MS pathology. In apparent contradiction to these results, a role and in animal models of MS, cardiomyopathy and stroke, of B-crystallin in positive modulation of the NF-B signal- and treatment with recombinant B-crystallin has shown ing pathway has recently been demonstrated. In this report, promise to reduce inflammatory responses, improving the B-crystallin protected muscle myoblasts from TNF-- outcome in experimental animal models of neuro- induced cytotoxicity through up-regulation of Bcl2 [71]. inflammation and stroke. However, it has not been deter- Further investigations are warranted to understand the mined how B-crystallin is released from cells during patho- mechanisms by which B-crystallin alters inflammatory logical conditions in vivo, and the mechanisms by which responses, and subsequently angiogenesis, in different patho- extracellular B-crystallin limits inflammation have not been logical conditions. fully investigated. Notably, release of B-crystallin from retinal pigment epithelial cells has recently been shown to be B-CRYSTALLIN - A TARGET FOR CANCER mediated by a non-classical exosomal pathway [80], but it THERAPY? remains to be examined if this pathway operates also in other cell types and conditions. Another important question is to B-crystallin expression can be induced both by cell understand in which context B-crystallin may act as a stress and /or transformation-dependent aberrant expression chaperone for pro-angiogenic growth factors such as FGF-2 or function of transcription factors in tumor cells. Therefore, and VEGF. Finally, the potential usefulness of targeting B- it is not surprising that high levels of B-crystallin have been crystallin in cancer therapy is still not clear. Will targeting of 44 Current Angiogenesis, 2012, Vol. 1, No. 1 Dieterich and Dimberg an intrinsic regulator of angiogenesis, such as B-crystallin, [17] Ecroyd H, Meehan S, Horwitz J, et al. Mimicking phosphorylation decrease tumor resistance to anti-angiogenic treatment? of alphaB-crystallin affects its chaperone activity. Biochem J 2007; 401(1): 129-41. What would the effects be on tumor aggressiveness and me- [18] Ahmad MF, Raman B, Ramakrishna T, Rao Ch M. Effect of tastasis? And finally, would the anti-angiogenic and anti- phosphorylation on alpha B-crystallin: differences in stability, tumor effects be counteracted by increased inflammation in subunit exchange and chaperone activity of homo and mixed the tumor microenvironment? oligomers of alpha B-crystallin and its phosphorylation-mimicking mutant. J Mol Biol 2008; 375(4): 1040-51. [19] Benesch JL, Ayoub M, Robinson CV, Aquilina JA. Small heat CONFLICT OF INTEREST shock protein activity is regulated by variable oligomeric substructure. J Biol Chem 2008; 283(42): 28513-7. None. [20] Vicart P, Caron A, Guicheney P, et al. A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related ACKNOWLEDGEMENTS myopathy. Nat Genet 1998; 20(1): 92-5. [21] Bova MP, Yaron O, Huang Q, et al. Mutation R120G in alphaB- We apologize to colleagues in the field whose work crystallin, which is linked to a desmin-related myopathy, results in could not be cited due to space constraints. 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Received: December 22, 2011 Revised: January 24, 2012 Accepted: February 27, 2012