Mervyn J. Merrilees¹ and Thomas N. Wight

Versican is a chondroitin sulfate (CS) proteoglycan present in the interstitial space of all tissues and is notably elevated during early development, where it plays a key role in organogenesis, and later in numerous pathologies, often those affecting major organs such as vessels, lung and skin [1]. Accumulating evidence indicates that versican is of critical importance in chronic vascular diseases including atherosclerosis, aneurysms and restenosis [2]. Versican is also elevated in many types of cancers and can directly influence the metastatic potential of tumors [3-6]. Versican increases in the airways and parenchyma of lungs affected by emphysema and chronic obstructive pulmonary disease (COPD), and is elevated in some rare lung diseases such as lymphangioleiomyomatosis (LAM) [7]. Recent evidence indicates it is a central player in inflammation, which broadens its importance to acute events in tissue repair. Versican is thus emerging as a potential target promising wide therapeutic benefits.

Versican is highly interactive with versatility reflected in its name. The versican gene and protein follow a domain template with an amino-terminal globular domain (G1) that binds hyaluronan, a carboxy-terminal globular domain (G3) similar to the selectin family of proteins with a C-type lectin adjacent to two epidermal growth factor domains and a complement region, and a middle region encoded by two large exons specifying CS attachment regions [8].

Versican’s innate complexity is further reflected by alternative splicing of exons 7 and 8 that encode the glycosaminoglycan (GAG) attachment domains, giving rise to V0, V1, V2, and V3 variants. V3 has neither of these exons, so is without GAG chains, but paradoxically has a demonstrated ability to dramatically influence structure and composition of the extracellular matrix (ECM) in potentially beneficial ways [2,9]. In addition to variation in core protein size due to splicing, the CS chains also vary in size (25 to 80kDa) as does composition, depending on tissue source and stimuli, with variation in the ratio of chondroitin-6-sulfate to chondroitin-4-sulfate. Expression of the versican gene is regulated by a promoter harboring a typical TATA box, β-catenin/TCF response elements and potential binding sites for transcription factors such as SP1, p53, AP1, AP2, CCAAT enhancer protein, and cAMP-responsive elements [10,11]. Regulation of core protein synthesis can be at transcriptional and posttranscriptional levels.

Studies on vessels have contributed most to our understanding of the roles of versican in pathogenesis. It is present in early intimal thickenings, and is elevated in human vessels susceptible to atherosclerosis. It contributes to intimal expansion and intimal progression and conversely its degradation is accompanied by lesion regression [12]. It is present around the core of large lesions and there is good evidence that its CS chains interact with and retain LDL in the vessel wall; it is thus central to the response-to-retention hypothesis of atherogenesis. Binding sites for versican have been identified in human apoprotein B of LDL, and expression of mutated apoB with defective binding dramatically ameliorates development of lesions in animals fed a high fat diet [13].

Versican is also elevated in aneurysms and restenosis, two conditions in which there is substantial remodelling of tissues, including marked changes to elastic fibers for which the GAG-containing variants are negative regulators. Assembly of elastic fibers, which involves incorporation of tropoelastin onto the microfibrillar scaffold, a process mediated via cell surface elastin binding protein, is inhibited by CS. On the other hand, over-expression of V3 lacking CS chains decreases the tissue content of the CS-containing versican variants and markedly enhances elastogenesis and the assembly of elastic fibers in the matrix [14]. Alternatively, decreasing CS-containing versican variants by over-expression of V3 or application of versican antisense sequences similarly promotes elastogenesis [15]. V3 also increases deposition of elastin by skin fibroblasts, and has been utilized in vitro to correct the elastin deficiency that characterizes Costello syndrome [16]. This inverse relationship between versican and elastin is also present in lungs affected by LAM and COPD, and in the latter versican levels correlate negatively with lung function measures such as FEV1 [7]. This opens up the possibility for V3 or versican antisense interventions in these respiratory diseases.

Recently, we demonstrated that manipulation of versican levels in the repair tissue (neointima) of balloon-injured arterial wall, through over-expression of V3, dramatically remodels the ECM, resulting in a compact, elastin-enriched and layered neointima that is not only resistant to cholesterol deposition, but also to macrophage ingress [9]. The reduced deposition of cholesterol is consistent with versican’s role in retaining lipoproteins, but the additional advantage of reducing inflammation broadens the benefits of targeting the CS-containing versican variants.

Several lines of evidence point to versican as an important mediator of inflammation, as well as being a prometastatic agent. Versican V1 stimulates proinflammatory cytokines tumor necrosis factor- (TNF-) and interleukin 6 through the Toll 2 receptor [4], and mediates leukocyte aggregation through binding to P-selectin glycoprotein ligand-1 [17]. Further, versican promotes adhesion of monocytes in a hyaluronan-dependent manner, an interaction that that is significantly reduced in matrices in which versican has been reduced by versican antisense or by V3-expression [9], versican shRNA [6] or blocked by an antibody to versican [18]. Such findings highlight a potential therapeutic application for targeting versican as anti-inflammatory therapy in the treatment of diseases such as atherosclerosis and cancer.

The association between versican and metastasis is clearly illustrated by breast and prostate cancers, where elevated levels of peritumoral stromal versican correlate positively with tumor grade and invasiveness [5]. Experimental evidence demonstrating that versican is central to metastasis comes from studies on Lewis lung carcinoma (LLC) cells. Versican from LLC cells directly mediates metastasis through activation of Toll-like receptor 2 on macrophages which then up-regulate TNF-. Elevated expression of versican is seen in myeloid progenitor cells in metastatic lungs in a mouse model of breast cancer and in the metastatic lungs of human patients with breast cancer [3]. Knockdown of versican in the bone marrow progenitor cells significantly impairs lung metastasis in this breast cancer mouse model. In addition, lung metastases that develop in a murine model of bladder cancer could be controlled by targeting tumor expression of versican [6].

In summary, the GAG-containing versican variants are emerging as potential targets for therapeutic intervention across a variety of conditions where tissues are undergoing growth and remodelling associated with expansion, degradation and inflammation. Interestingly and seemingly paradoxically, the variant without GAG chains, V3, acts to counter the changes induced by its parent molecules, dampening growth and inflammation and promoting formation of elastin-rich and differentiated tissues.

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References

1. Wight TN: Versican: a versatile extracellular matrix proteoglycan in cell biology. Curr Opin Cell Biol 2002, 14:617-623.
2. Wight TN, Merrilees MJ: Proteoglycans in atherosclerosis and restenosis: key roles for versican. Circ Res 2004, 94:1158-1167.
3. Gao D, Joshi N, Choi H, Ryu S, Hahn M, Catena R, Sadik H, Argani P, Wagner P, Vahdat LT, et al.: Myeloid progenitor cells in the premetastatic lung promote metastases by Inducing mesenchymal to epithelial transition. Cancer Res 2012, In press.
4. Kim S, Takahashi H, Lin WW, Descargues P, Grivennikov S, Kim Y, Luo JL, Karin M: Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature 2009, 457:102-106.
5. Ricciardelli C, Sakko AJ, Ween MP, Russell DL, Horsfall DJ: The biological role and regulation of versican levels in cancer. Cancer Metastasis Rev 2009, 28:233-245.
6. Said N, Sanchez-Carbayo M, Smith SC, Theodorescu D: RhoGDI2 suppresses lung metastasis in mice by reducing tumor versican expression and macrophage infiltration. J Clin Invest 2012, In press
7. Merrilees M, Ching P, Beaumont B, Hinek A, Wight T, Black P: Changes in elastin,elastin binding protein and versican in alveoli in chronic obstructive pulmonary disease. Respir Res 2008, 18:41-50.
8. Zimmermann DR, Ruoslahti E: Multiple domains of the large fibroblast proteoglycan, versican. EMBO J 1989, 8:2975-2981.
9. Merrilees MJ, Beaumont BW, Braun KR, Thomas AC, Kang I, Hinek A, Passi A, Wight TN: Neointima formed by arterial smooth muscle cells expressing versican variant v3 is resistant to lipid and macrophage accumulation. Arterioscler Thromb Vasc Biol 2011, 31:1309-1316.
10. Domenzain-Reyna C, Hernandez D, Miquel-Serra L, Docampo MJ, Badenas C, Fabra A, Bassols A: Structure and regulation of the versican promoter: the versican promoter is regulated by AP-1 and TCF transcription factors in invasive human melanoma cells. J Biol Chem 2009, 284:12306-12317.
11. Rahmani M, Wong BW, Ang L, Cheung CC, Carthy JM, Walinski H, McManus BM: Versican: signaling to transcriptional control pathways. Can J Physiol Pharmacol 2006, 84:77-92.
12. Kenagy RD, Plaas AH, Wight TN: Versican degradation and vascular disease. Trends Cardiovasc Med 2006, 16:209-215.
13. Borén J, Olin K, Lee I, Chait A, Wight TN, Innerarity TL: Identification of a principal proteoglycan binding site in LDL: A single point mutation in apo B-100 severely affects proteoglycan interaction without affecting LDL receptor binding. J. Clin. Invest. 1998, 101:2658-2664.
14. Merrilees MJ, Lemire JM, Fischer JW, Kinsella MG, Braun KR, Clowes AW, Wight TN: Retrovirally mediated overexpression of versican v3 by arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in vitro and in neointima after vascular injury. Circ Res 2002, 90:481-487.
15. Huang R, Merrilees MJ, Braun K, Beaumont B, Lemire J, Clowes AW, Hinek A, Wight TN: Inhibition of versican synthesis by antisense alters smooth muscle cell phenotype and induces elastic fiber formation in vitro and in neointima after vessel injury. Circ Res 2006, 98:370-377.
16. Hinek A, Braun KR, Liu K, Wang Y, Wight TN: Retrovirally mediated overexpression of versican v3 reverses impaired elastogenesis and heightened proliferation exhibited by fibroblasts from Costello syndrome and Hurler disease patients. Am J Pathol 2004, 164:119-131.
17. Zheng PS, Vais D, Lapierre D, Liang YY, Lee V, Yang BL, Yang BB: PG-M/versican binds to P-selectin glycoprotein ligand-1 and mediates leukocyte aggregation. J Cell Sci 2004, 117:5887-5895.
18. Potter-Perigo S, Johnson PY, Evanko SP, Chan CK, Braun KR, Wilkinson TS, Altman LC, Wight TN: Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion. Am J Respir Cell Mol Biol 2010, 43:109-120.

 
Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Posted in Cell Biology

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Recent studies have raised the intriguing possibility that losartan, the prototypical angiotensin II (AngII) receptor type 1 (AT1r) blocker (ARB), should be used as a prophylactic drug akin to aspirin and statins. Other reports however have questioned whether losartan is the real thing or is this a case of unbridled enthusiasm fueling yet another bubble. While only time will tell, it is worth reviewing the pros and cons regarding losartan’s broad therapeutic potential. Since FDA approval in 1995, ARBs have been widely used to treat hypertension, cardiac hypertrophy and renal disease owing to their selective antagonism of AngII-induced arterial muscle contraction, sympathetic pressor mechanisms and aldosterone release [1] Losartan was also shown to reduce AT1r-induced TGFβ activity in a mouse model of human cardiomyopathy [2]. This last observation prompted investigators to test losartan in several experimental models of tissue fibrosis, as TGFβ is a potent determinant of extracellular matrix (ECM) deposition and remodeling. The studies eventually converged on the discovery that losartan also can mitigate thoracic aortic aneurysm (TAA) in Marfan syndrome (MFS), a common connective tissue disease associated with mutations in the ECM protein fibrillin-1 [3]. This seminal finding was welcomed by the medical community with understandable enthusiasm because it represented the first pharmacological treatment of a condition whose treatment options were thought to be restricted to gene or stem cell therapy.

Management of TAA progression in MFS currently relies on agents that lower blood pressure to delay aortic dilation and on elective aortic root replacement to prevent dissection and rupture. Aside from imparting structural properties to tissues, fibrillin-1 regulates cell performance by modulating extracellular bioavailability of latent TGFβ complexes. Consistent with the latter function, affected tissues from MFS patients and MFS mice display promiscuous TGFβ signaling, conceivably as the result of improper regulation of TGFβ bioavailability. Thus the beneficial impact of losartan treatment on vessel dilation and tissue degeneration may reflect respectively the drug’s anti-hypertensive and anti-TGFβ signaling properties, which are both highly desirable outcomes in MFS patients. Not only did losartan normalize aortic dilation and aortic wall architecture in MFS mice (Fbn1^C1039G/+ mice), but a retrospective study of a small cohort of severely affected MFS children revealed an appreciable benefit of using ARBs to slow aortic root growth (Habashi 2006; Brooke 2008). Additionally, losartan was reported to improve skeletal muscle abnormalities of MFS mice and mice with either Duchene muscular dystrophy (DMD) or age-related sarcopenia [4;5]. Furthermore the finding that losartan significantly reduces tumor growth of AT1r over-expressing ERBB2-negative breast cancer xenografts implied potential applications in cancer therapy [6]. The finding that improper TGFβ signaling is central to several pathological conditions therefore supports the notion that losartan may have pharmacological benefits beyond those originally envisioned, which presumably reflect the multiple biological functions of AT1r signaling (1;7]. However there are data advocating an alternative view. 

In contrast to full TAA rescue in Fbn1^C1039G/+ mice, losartan treatment only delays aortic dissection and rupture in Fbn1^mgR/mgR mice, which have a more substantial fibrillin-1  abnormality and are more seriously affected than Fbn1^C1039G/+ mice (Baxter B.T., personal communication). Losartan’s ability to blunt maladaptive aortic tissue remodeling in MFS mice therefore appears to be inversely proportional to the severity of the ECM defect, which determines the extent of both tissue impairment and TGFβ dysregulation. It follows that different MFS patients may respond differently to ARB treatment depending on the nature of the FBN1 mutation and genetic modifiers of ECM composition and TGFβ and/or AT1r activity. Additional studies have questioned the broad applicability of losartan and other ARBs. First, the original finding that TGFβ antagonism improves multiple manifestations in MFS mice was later counterbalanced by losartan’s inability to normalize skeletal abnormalities in these mutant mice [3;4;8]. Second, Bish et al. [9] recently showed that losartan treatment of DMD mice improves cardiomyopathy but not skeletal muscle degeneration, as previously reported [4]. With respect to losartan and cancer therapy, decreasing TGFb signaling might in principle ameliorate the fibrosis associated with some tumors but might also promote growth of early neoplasms, as TGFβ is a potent tumor suppressor.

In spite of controversial findings, the above studies have brought us closer to developing therapeutic strategies for MFS that are based on experimental evidence and mechanistic insights. Irrespective of being either a proximal or distal effector of TAA progression, TGFβ has proven to be a viable biological target in a genetic disease with few other therapeutic options.  This in turn provides the justification and means to search for highly specific small molecule inhibitors that will circumvent potential problems of losartan treatment, such as optimal dosage, genetic variability in drug response, long-term side effects etc. Even if losartan is not the anticipated panacea for MFS and other diseases, there is still significant merit in using AT1r antagonism to dissect pathogenic mechanisms and identify more suitable biological targets. Last but not least, the studies of MFS pathogenesis have underscored the physiological importance of an understudied research topic, namely ECM-mediated control of growth factor bioavailability. It seems therefore reasonable to conclude that perhaps we should embrace rather than repudiate controversial and paradoxical findings for they are the dialectic engine that propels scientific progress.

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References

1. Burnier M, Wuerzner G. (2011) Pharmacokinetic evaluation of losartan. Expert Opin. Metab. Toxicol. 7:643-649.
2. Lim DS, Lutucuta S, Bachireddy P, Youker K, Evans A, Roberts R, Marian AJ. (2001) Angiotensin II blockade reverses myocardial fibrosis in a transgenic mouse model of human hypertrophic cardiomyopathy. Circulation 103:789-791.
3. Brooke BS, Habashi JP, Judge DP, Patel N, Loeys B, Dietz HC 3rd. (2008) Angiotensin II blockade and aortic-root dilation in Marfan’s syndrome. N Engl. J. Med. 358:2787-2795.
4. Cohn RD, van Erp C, Habashi JP, Soleimani AA, Klein EC, Lisi MT, Gamradt M, ap Rhys CM, Holm TM, Loeys BL, Ramirez F, Judge DP, Ward CW, Dietz HC. (2007) Angiotensin II type 1 receptor blocade attenuates TGF--induced failure of muscle regeneration in multiple myopathic states. Nat. Med. 13:204-210.
5. Burks TN, Andres-Mateos E, Marx R, Mejias R, Van Erp C, Simmers JL, Walston JD, Ward CW, Cohn RD. (2011) Losartan restores skeletal muscle remodeling and protects against disuse atrophy in sarcopenia. Sci. Transl Med.3:82ra37.
6. Rhodes DR, Ateeq B, Cao Q, Tomlins S.A., Mehra R., Laxman B., Kalyana-Sundram S, Lonigro RJ, Helgeson BE, Bhojani MS, Rehemtulla A, Kleer CG, Hayes DF, Lucas PC, Varambally S, Chinnaiyan AM. (2009) AGTR1 overexpression defines a subset of breast cancer and confers sensitivity to losartan, an AGTR1 antagonist. Proc. Natl. Acad. Sci. 106:10284-10289.
7. Mederos y Schnitzler M, Storch U, Gudermann T. (2011) AT1 receptors as mechanosensors. Curr. Opin. Pharmacol. 11:112-116.
8. Nistala H, Lee-Arteaga S, Carta L, Cook JR, Smaldone S, Siciliano G, Rifkin AN, Dietz HC, Rifkin DB, Ramirez F. (2010) Differential effects of alendronate and losartan therapy on osteopenia and aortic aneurysm in mice with severe Marfan syndrome. Hum. Mol. Genet. 19:4790-4798.
9. Bish LT, Yarchoan M, Sleeper MM, Gazzara JA, Morine KJ, Acosta P, Barton ER, Sweeney HL (2011) Chronic losartan administration reduces mortality and preserves cardiac but not skeletal muscle function in dystrophic mice. PLoS One 6:e20856.

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Posted in Pharmacology

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Glycosaminoglycans (GAGs), linear macromolecular heteropolysaccharides consisting of disaccharide repeating units, are classified in several types. Hexuronic acid based GAGs include heparin and heparan sulfate (HS), which are glucosamine containing sulfated GAGs, chondroitin and dermatan sulphates (CS and DS), based on galactosamine, and hyaluronan which is a glucosamine based non-sulfated GAG..

Keratan sulfate is a non hexuronic acid galactose containing sulfated GAG. They were originally defined as inert “glue” surrounding the cells and were thus positioned on the side-track of “cutting-edge” research efforts for decades. During the last years, however, a huge leap in the field has been accomplished and these macromolecules are now recognized as essential players in critical biological processes regulating cellular properties; tissue development and remodelling; homeostasis; and disease progression

The unique structural characteristics at the level of sulfation within the GAG chains are closely related with their diverse functions. The evidence that GAGs have a key role in various pathological conditions, have led to the conclusion that understanding the changes in GAG expression and fine structure that occur in disease may lead to opportunities to develop innovative and selective therapies (Reviewed by [1]). This has initiated numerous and novel approaches to exploit pluripotent characteristics of GAGs in the ongoing battle against disease.

Acknowledgement of GAGs as potential therapeutic agents dates from the beginning of 19^th century. The highly sulfated, free GAG, heparin was discovered in 1916 to have potent anticoagulation properties, whereas the first clinical trials in 1941, introduced heparin as a pioneer drug in the field of GAG therapeutics. The extensive use of this wonder agent, has promoted the progress and development of vascular and cardiac surgery, the extracorporeal circulation, the haemodialysis, the organ transplantation and the treatment and prevention of arterial and venous thromboembolism. This impressive track history is based on heparins’ ability to interact with anticoagulant proteins like antithrombin and heparin cofactor II, promoting their activation and increasing their ability to inhibit thrombin.

Frequent use of heparin in the treatment of cancer-associated thromboembolism initially pointed out its anti-cancer potential. Accumulating clinical evidence indicates that cancer patients treated with unfractionated and low-molecular weight heparin (LMWHP) survive longer than patients treated by other anticoagulants, especially patients in the early stage of the disease. The non-anticoagulant activity of heparin on metastasis includes the ability to inhibit cell-cell-interaction through blocking of P- and L-selectin, to inhibit the extracellular matrix enzyme heparanase, to modulate the binding of growth factors involved in epithelial to mesenchymal transition during tumour invasion and to inhibit angiogenesis [2]. Notably, the inhibition of melanoma cell adhesion and migration by LMWHP via the PKCa/JNK signaling axis affecting actin cytoskeleton changes opens another area in GAG therapeutics for the future [3].

The extraordinary possibilities of GAG applications in targeted disease treatment are further illustrated by the ability of heparin to regulate processes correlated with inflammation. Heparin participates in the regulation of the inflammatory response by inhibiting the influx of neutrophils into certain tissues and attenuating T-cell trafficking, partly by an inhibitory effect on the heparanase secreted by T-cells. In the lung, it has been suggested that inhibition of the interaction between pro-inflammatory cytokines and membrane-associated GAGs by heparin may provide a mechanism for inducing clinically useful immunosuppression (reviewed [4]).

Structurally similar to heparin, HS which in the cellular milieu is bound into proteoglycans (PGs) facilitates both angiogenesis and the activity of the HS-cleaving heparanase. The HS side chains of PGs present in basal membrane contribute not only to storing and preserving the biological activity of various HS-binding cytokines and growth factors, but also in presenting them in a more “active conformation” to their cognate receptors. Abnormal expression or deregulated function of these PGs affect cancer and angiogenesis, and are critical for the evolution of the tumour microenvironment [5]. Furthermore, GAGs as binding partners for matrix metalloproteinases and protease inhibitors, regulate the proteolytic microenvironment of tumours, thus modulating metastatic spread.

Heparan sulfate mimetics, that have been developed to inhibit these processes, are currently undergoing formal preclinical development as a novel treatment for advanced cancer [6]. Heparan sulfate membrane PGs may also be a scaffold that facilitates the interaction of intracellular pathogens with secondary receptors that mediate host cell entry, a key step in the infection process. Consistent with this mechanism, application of HS or heparin as well as modulation of host cell membrane HS inhibits microbial attachment and entry [7].

Chondroitin and dermatan sulphates also have intriguing biological activities, which in turn should help the development of CS/DS-based therapeutics (reviewed by [8]). In the milieu of cancer deregulation, the observation that CS is overexpressed in several highly metastatic tumours led to the suggestion that CS may well be used as a target for the selective delivery of anticancer drugs by polyethylene glycol-coated liposomes.

Hyaluronan matrices are ubiquitous in normal and pathological biological processes. Indeed, many cell stress responses initiate the synthesis of a monocyte-adhesive hyaluronan extracellular matrix, which forms a central focus for subsequent inflammatory processes that are modulated by the dialogue between the matrix and the inflammatory cells [9].

The specific physicochemical properties of GAGs allow these molecules to support and re-establish structural tissue homeostasis which resulted in their extensive use in orthopaedic clinical practice as well as in reconstructive and cosmetic surgery. Chondroitin sulphate belongs to the oral symptomatic slow-acting drugs for the treatment of osteoarthritis. The evidence for clinical efficacy of oral CS as a drug comes from sets of clinical trials which document its good tolerability and safety aspects [10]. Moreover, HA has been extensively used in both reconstructive and cosmetic surgery due to its effectiveness, ease of administration, and safety profile.

The endeavours of the “wonder” drug heparin are well known. Encouraging studies, briefly outlined above, strongly indicate the potential use of GAGs as potent therapeutic agents in this promising field of targeted disease therapy.

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References

1. Theocharis AD, Skandalis SS, Tzanakakis GN, Karamanos NK: Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting. FEBS J 2010, 277:3904-3923.
2. Kozlowski EO, Pavao MS: Effect of sulfated glycosaminoglycans on tumor invasion and metastasis. Front Biosci (Schol Ed) 2011, 3:1541-1551.
3. Chalkiadaki G, Nikitovic D, Katonis P, Berdiaki A, Tsatsakis A,Kotsikogianni I, Karamanos NK, Tzanakakis GN: Low molecular weight heparin inhibits melanoma cell adhesion and migration through a PKCa/JNK signaling pathway inducing actin cytoskeleton changes. Cancer Lett 2011, 312:235-244.
4. Souza-Fernandes AB, Pelosi P, Rocco PR: Bench-to-bedside review: the role of glycosaminoglycans in respiratory disease. Crit Care 2006, 10:237.
5. Iozzo RV, Zoeller JJ, Nystro¨m A: Basement membrane proteoglycans: modulators Par Excellence of cancer growth and angiogenesis. Mol Cells 2009, 27:503-513.
6. Dredge K, Hammond E, Davis K, Li CP, Liu L, Johnstone K, Handley P, Wimmer N, Gonda TJ, Gautam A et al.: The PG500 series: novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy. Invest New Drugs 2010, 28:276-283.
7. Li Y, Sun JF, Cui X, Mani H, Danner RL, Li X, Su JW, Fitz Y, Eichacker PQ: The effect of heparin administration in animal models of sepsis: a prospective study in Escherichia colichallenged mice and a systematic review and metaregression analysis of published studies. Crit Care Med 2011, 39:1104-1112.
8. Yamada S, Sugahara K: Potential therapeutic application of chondroitin sulfate/dermatan sulfate. Curr Drug Discov Technol 2008, 5:289-301.
9. Wang A, de la Motte C, Lauer M, Hascall V: Hyaluronan matrices in pathobiological processes. FEBS J 2011, 278:1412-1418.
10. Uebelhart D: Clinical review of chondroitin sulfate in osteoarthritis. Osteoarthritis Cartilage (Suppl. 3):2008:S19-S21.

DOI: 10.1016/j.coph.2011.12.003

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Posted in Pharmacology

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Heparin is a complex  polysaccharide-based anticoagulant drug that is essential for the practice of modern medicine [1]. Used in extracorporeal therapies, such as kidney dialysis, in the treatment of coagulation disorders, such as deep vein thrombosis (DVT), and to passivate the surfaces of indwelling devices, such as catheters, a heparin-based product is the first choice whenever blood clotting needs to be prevented or controlled.

There are three forms of heparin, unfractionated heparin (molecular weight average MW_avg ~15,000), low molecular weight heparin (LMWH, MW_avg ~6,000), and ultra low molecular weight heparin (ULMWH, MW_avg <2,000) [1-3]. The most widely used form is unfractionated heparin, a century-old drug prepared from animal tissues. Despite its widespread use it has several limitations including required intravenous administration and side effects such as heparin induced thrombocytopenia (HIT) and bleeding. In the 1990s, LMWHs were introduced and they have successfully captured a large share of the market becoming the anticoagulant of choice to treat DVTs. LMWHs are prepared from animal-sourced unfractionated heparin through controlled depolymerization. These have the major advantage of being subcutaneously bioavailable and have a longer half-life allowing their outpatient use and/or self-administration. A major disadvantage of LMWHs is that, unlike unfractionated heparin, their anticoagulant effect is not readily reversible, thus increasing the risk for bleeding due to overdosing. The last decade has brought us ULMWHs (i.e., fondaparinux), first synthesized by Sinaÿ, Choay and coworkers [4], which have well controlled pharmacokinetics/pharmacodynamics, have no viral or prion impurities (possible in animal-sourced materials), are subcutaneously bioavailable, and are manufactured under current good manufacturing process (cGMP). Unfortunately, these agents are very expensive and like LMWHs are not readily reversible.

In 2007-2008, there was a heparin crisis that resulted from contaminated batches of heparin and LMWH entering the marketplace [5]. Severe side effects (i.e., a rapid drop in blood pressure), some leading to death, were ascribed to batches of contaminated unfractionated heparin imported from China. This crisis led to the recall of much of the heparin on the market and could have been a much greater problem had there not been sufficient amounts of non-contaminated product to meet the needs of dialysis and surgery patients. The contamination was traced to an adulteration of the crude heparin precursor with an oversulfated chondroitin sulfate somewhere in the process between the slaughterhouse where heparin was collected from pig intestines and the pharmaceutical manufacturing site. While ULMWH was not contaminated, it could not be relied on to alleviate this crisis because of its high cost, difficulty to produce in sufficient quantities to meet worldwide needs, and limited utility for kidney dialysis. The inspection of foreign suppliers and upgrading the pharmacopeial monographs has reduced the likelihood of a similar crisis in the future but the increased demand on heparin, as modern medicine is applied to more of the world, and the limited number of pigs (1 pig provides ~3 doses of unfractionated heparin or ~1 dose of LMWH) pose constraints on the supply of this critical drug. Since the crisis, the cost of heparin active pharmaceutical ingredient (API) has increased 10-fold.

Heparin is biosynthesized within the Golgi organelles of mast cells that are most commonly found in intestine, lung, liver and skin of higher animals [1]. Heparin biosynthesis, elucidated through the elegant work of Lindahl and coworkers [6], starts with the building of its linear polysaccharide backbone (even some bacteria are capable of this step) through the synthase-catalyzed alternating addition of two UDP-sugars. Backbone sugars are enzymatically N-deacetylated, N-sulfonated, O-sulfonated and epimerized at selected locations, affording the highly sulfated heparin. The enzymes involved in heparin biosynthesis are known and many have been cloned in the past decade. These enzymes have been largely used as tools by biochemists studying heparin structure and biosynthesis [7]. While much is now understood about their in vitro activity and specificity, little is known about how they are regulated and controlled in vivo in the Golgi. It is clearly understood, however, that the control of these enzymes could offer a great potential in the preparation of synthetic unfractionated heparin, LMWH and ULMWH. Moreover, it is also possible to consider the preparation of designer heparins [8] with reduced side effects (i.e., HIT), more defined physical, chemical, biological and pharmacological properties.  

In 2007, our laboratories chemoenzymatically prepared a small amount of bioengineered heparin from an E. coli-derived polysaccharide [9]. Others, including Kuberan and Rosenberg [10], Lindahl and Casu [11], and DeAngelis [12] had also used similar approaches to prepare other heparin-like polysaccharides and oligosaccharides. Over the past 4 years, we have been aggressively examining the possibility of preparing sufficient quantities of a bioengineered, unfractionated heparin to meet the global supply needs (~100 tons/yr.) as a generic version of heparin [5,13]. The process begins with an E. coli fermentation to prepare polysaccharide, chemical de-N-acetylation, N-sulfonation a process that is now moving to the kilogram scale. The use of recombinant O-sulfotransferases and C5-epimerase results in a bioengineered heparin that closely resembles the chemical and biological properties of heparin. One critical component of this process is to reduce the cost of the sulfotransferase cofactor and sulfo group-donor PAPS, making up half the product mass, is being addressed by improved PAPS production and cofactor recycling [14, 15].

Recently, our laboratories have announced a success in preparing an ULMWH, similar to fondaparinux, using a related chemoenzymatic process (3). Instead of preparing the oligosaccharide backbone through fermentation, it is enzymatically synthesized by iterative addition of UDP-sugars. This is again followed by the use of recombinant O-sulfotransferases and C5-epimerase to afford a pure ULMWH in over a 100-fold higher yield than possible using chemical synthesis. The two ULMWH constructs prepared showed comparable pharmacological properties as fondaparinux. It remains to be seen whether this chemoenzymatic process can be commercialized to afford new ULMWHs for regulatory agency approval.

In summary, heparin-based therapeutics are essential to modern medicine and will remain an important class of drugs for the foreseeable future. Modern biotechnological methods are now available to bring more advanced manufacturing process for heparins from the extractive methods developed in the early 20^th century and the chemically synthetic methods of the late 20^th century into the new millennium. The world is ready and waiting for the next generation of synthetic heparin.

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References

1. Linhardt RJ: Heparin: structure and activity. J Med Chem 2003, 46:2551-2554.
2. Linhardt RJ, Gunay NS: Production and chemical processing of low molecular weight heparins. Semin Thrombos Hemostas 1999, 25:5-16.
3. Xu Y, Masuko S, Takieddin M, Xu H, Liu R, Jing J, Mousa S, Linhardt RJ, Liu J: Chemoenzymatic synthesis of structurally homogeneous ultra-low molecular weight heparins. Science 2011, 334:498-501.
4. Sinay¨ P, Jacquinet J-C, Petitou M, Duchaussoy P, Lederman I, Choay J, Torri G: Total synthesis of a heparin pentasaccharide fragment having high affinity for antithrombin III. Carbohydr Res 1984, 132:C5-C9.
5. Liu H, Zhang Z, Linhardt RJ: Lessons learned from the contamination of heparin. Nat Prod Rep 2009, 26:313-321.
6. Lindahl U, Kusche-Gullberg M, Kjelle´ n L: Regulated diversity of heparan sulfate. J Biol Chem 1998, 273:24979-24982.
7. Esko JD, Selleck SB: Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem 2002,71:435-471.
8. Peterson S, Frick A, Liu J: Design of biologically active heparan sulfate and heparin using an enzyme-based approach. Nat Prod Rep 2009, 26:610-627.
9. Zhang Z, McCallum SA, Xie J, Nieto L, Corzana F, Jime´ nez-Barbero J, Chen M, Liu J, Linhardt RJ: Solution structures of chemoenzymatically synthesized heparin and its precursors. J Am Chem Soc 2008, 130:12998-13007.
10. Kuberan B, Lech MZ, Beeler DL, Wu ZL, Rosenberg RD: Enzymatic synthesis of antithrombin III-binding heparan sulfate pentasaccharide. Nat Biotechnol 2003, 21:1343-1346.
11. Lindahl U, Li JP, Kusche-Gullberg M, Salmivirta M, Alaranta S, Veromaa T, Emeis J, Roberts I, Taylor C, Oreste P et al.: Generation of ‘‘neoheparin’’ from E. coli K5 capsular polysaccharide. J Med Chem 2005, 48:349-352.
12. Kane TA, White CL, DeAngelis PL: Functional characterization of PmHS1, a Pasteurella multocida heparosan synthase. J Biol Chem 2006, 281:33192-33197.
13. Wang Z, Yang B, Zhang Z, Ly M, Takieddin M, Mousa S, Liu J, Dordick JS, Linhardt RJ: Control of the heparosan Ndeacetylation leads to an improved bioengineered heparin. Appl Microbiol Biotechnol 2011, 91:91-99.
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DOI: 10.1016/j.coph.2011.12.002

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Posted in Pharmacology

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Decorin and biglycan, the two best studied members of the small leucine-rich proteoglycan (SLRP) family, have been implicated in regulating cancer growth and inflammation, respectively. Decorin expression is almost always suppressed by cancer cells but abundantly produced by activated stromal fibroblasts in the tumor microenvironment [1]. Often an inverse relationship exists between cancer growth and decorin expression, suggesting that decorin is an “endogenous guardian” from the matrix. The mechanism of decorin-evoked tumor repression is linked to its ability to potently induce the endogenous synthesis of p21, a key inhibitor of cyclin-dependent kinases. This is carried out by soluble decorin binding in a paracrine fashion to several receptor tyrosine kinases (RTKs) including the EGFR, IGF-IR and Met [2]. Thus, decorin is a natural RTK inhibitor and systemic delivery of recombinant decorin inhibits the growth of various tumor xenografts [3,4]. Currently, it is a matter of debate of how decorin exactly inactivates specific receptors, given the fact that RTKs are ubiquitously expressed. One explanation involves a hierarchical mode of receptor affinity insofar as dissociation constants range from ~1 nM in the case of Met [5] to ~90 nM for EGFR. Thus, it could be envisioned that decorin, by acting as a pan-RTK inhibitor, would target many different types of tumors that exhibit differential RTK binding affinities for decorin. In most cases analyzed thus far, decorin evokes a rapid and protracted internalization of both EGFR and Met via caveolar-mediated endocytosis, a process that often leads to silencing of the receptors. Indeed, decorin blocks several biological processes associated with Met activation, such as cell scatter, evasion and migration [5]. One of the cellular mechanisms affected by this matrix molecule is via downregulation of the non-canonical -catenin pathway. This leads to suppression of Myc, a downstream target of -catenin, culminating in Myc proteasomal degradation [6]. Since Myc is a “master regulator” which can affect up to 1,500 genes, it is not surprising to predict that novel functional roles for decorin will be discovered in the near future.

The other SLRP structurally related to decorin, i.e., biglycan, acts as a danger signal and triggers both innate and adaptive immune responses. Under physiological conditions, the ubiquitously expressed biglycan is sequestered in the extracellular matrix and is immunologically inert. Upon tissue stress or injury, resident cells secrete proteolytic enzymes, which degrade the extracellular matrix and thus liberate biglycan and fragments thereof. Soluble biglycan and some of its fragments interact with Toll-like receptor (TLR)-2 and TLR4. By activating TLRs, biglycan triggers the synthesis of various proinflammatory cytokines and chemoattractants, thereby recruiting macrophages into damaged areas in order to resolve the injury [7]. Thus, soluble biglycan acts as a danger signal, which initiates a rapid innate immune response without the need for de novo synthesis of “warning” molecules. In addition, upon stimulation with proinflammatory cytokines, resident cells and infiltrating macrophages synthesize full-length biglycan leading to the recruitment of additional macrophages, which are also capable of synthesizing and secreting biglycan [7]. This creates a feed-forward loop that leads to robust proinflammatory signaling. Moreover, biglycan is capable of clustering TLR2/4 with purinergic P2X7 receptors, thereby autonomously activating the NLRP3 inflammasome and secretion of mature IL-1 [8].

Besides recruiting macrophages, biglycan stimulates the TLR2/4-dependent synthesis of key chemoattractants for T and B lymphocytes and is thus also involved in the adaptive immune response. Biglycan specifically recruits B1 lymphocytes which are responsible for T cell-independent production of antibodies. This represents an early defense against pathogens, before the adaptive immune response is activated. The biological importance of these mechanisms has been shown in systemic lupus erythematosus (SLE), a prototypic autoimmune disease affecting mainly young women. In SLE, soluble biglycan stimulates the synthesis of autoantibodies and enhances recruitment of macrophages as well as T and B lymphocytes resulting in enhanced inflammation in target organs. Notably, biglycan attracts B cells to chronically inflamed non-lymphoid organs and promotes the development of tertiary lymphoid tissue and acceleration of disease [9].

Collectively, these findings shed new light on the mechanisms of sterile inflammation, which plays a key role in tissue repair and regeneration (e.g., wound healing), ischemia/reperfusion injury (e.g., myocardial infarction) and autoimmune diseases (e.g., rheumatoid arthritis, SLE) among others. There is emerging evidence that soluble biglycan is generated in non-pathogen-mediated inflammatory diseases and autonomously triggers sterile inflammation by orchestrating TLR2/4 and NLRP3 inflammasome signaling [9]. On the other hand, in pathogen-mediated inflammation, the affinity of biglycan to receptors sensing either gram-positive or gram-negative pathogens allows for enhancement of inflammation via a second TLR, which is not involved in pathogen sensing [10].

The SLRPs are emerging as powerful signaling molecules affecting both cancer growth and inflammation. Thus, because cancer and inflammation are closely linked, we envisage that SLRPs such as decorin and biglycan could potentially become valid natural therapeutic agents or target themselves. A novel emerging concept is that, upon release from the extracellular matrix, a “basically inert” matrix component can turn into either a tumor repressor or a pro-inflammatory danger signal, which can subsequently drive innate and adaptive immune responses. This concept will offer novel perspectives in designing new pharmacological agents for therapeutic interventions in cancer, inflammatory and autoimmune diseases.

Multiple signaling pathways evoked by decorin and biglycan regulating cancer growth and inflammation. For details see text.

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References

1. Iozzo RV, Sanderson RD: Proteoglycans in cancer biology, tumour microenvironment and angiogenesis J. Cell. Mol. Med. 2011, 15:1013-1031.
2. Goldoni S, Iozzo RV: Tumor microenvironment: modulation by decorin and related molecules harboring leucine-rich tandem motifs. Int. J. Cancer 2008, 123:2473-2479.
3. Seidler DG, Goldoni S, Agnew C, Cardi C, Thakur ML, Owens RA, McQuillan DJ, Iozzo RV:Decorin protein core inhibits in vivo cancer growth and metabolism by hindering epidermal growth factor receptor function and triggering apoptosis via caspase-3 activation. J. Biol. Chem. 2006, 281:26408-26418.
4. Hu Y, Sun H, Owens RT, Wu J, Chen YQ, Berquin IM, Perry D, O’Flaherty JT, Edwards IJ: Decorin suppresses prostate tumor growth through inhibition of epidermal growth factor and androgen receptor pathways.Neoplasia 2009, 11:1042-1053.
5. Goldoni S, Humphries A, Nystro¨m A, Sattar S, Owens RT, McQuillan DJ, Ireton K, Iozzo RV: Decorin is a novel antagonistic ligand of the Met receptor. J. Cell Biol. 2009, 185:743-754.
6. Buraschi S, Pal N, Tyler-Rubinstein N, Owens RT, Neill T, Iozzo RV:Decorin antagonizes Met receptor activity and downregulates b-catenin and Myc levels. J. Biol. Chem. 2010, 285:42075-4208
7. Schaefer L, Babelova A, Kiss E, Hausser H-J, Baliova M, Krzyzankova M, Marsche G, Young MF, Mihalik D, Go¨ tte M, Malle E, Schaefer RM, Gro¨ ne H-J: The matrix component biglycan is proinflammatory and signals through toll-like receptors 4 and 2 in macrophages. J. Clin. Invest. 2005, 115:2223-2233.
8. Babelova A, Moreth K, Tsalastra-Greul W, Zeng-Brouwers J, Eickelberg O, Young MF, Bruckner P, Pfeilschifter J, Schaefer RM, Gro¨ ne H-J, Schaefer L: Biglycan, a danger signal that activates the NLRP3 inflammasome via Toll-like and P2X receptors. J. Biol. Chem. 2009, 284:24035-24048.
9. Moreth K, Brodbeck R, Babelova A, Gretz N, Spieker T, Zeng-Brouwers J, Pfeilschifter J, Young MF, Schaefer RM, Schaefer L: The proteoglycan biglycan regulates expression of the B cell chemoattractant CXCL13 and aggravates murine lupus nephritis. J. Clin. Invest. 2010, 120:4251-4272.
10. Iozzo RV, Schaefer L: Proteoglycans in health and disease: novel regulatory signaling mechanisms evoked by the small leucine-rich proteoglycans. FEBS J. 2010, 277:3864-3875

DOI: 10.1016/j.gde.2011.12.002

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Posted in Genetics & Development 

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Antidepressants have a peculiar place in the public mind. Almost every family in the UK will have someone taking these drugs; yet, the media are often very hostile to them with disparaging terms such as “happy pills” used to cast aspersions on their efficacy and utility [1]. Moreover, claims that they may cause suicidal ideation particularly in young people have lead to warning labels in the USA. Yet, despite the repeated media criticism, the use of antidepressants continues to be widespread. So what are the reasons for this?

The first is that they are effective and the disorders they are used to treat are very common. Depression is predicted to be largest cause of disability in the world by 2020 [2] and is now exceeding that of cardiovascular disease in many western countries (making it number one there already). Moreover, several of the anxiety disorders are also in the top ten causes of disability, and these also respond very well to antidepressant treatment.

In their primary target of depression, the antidepressants are effective treatments of the acute phase with a number needed to treat (NNT) of about 6, which compares favourably with treatments in other branches of medicine. However, when used in the long term to prevent recurrence of depression, they become even more effective with an NNT of 3 [3]. This makes them one of the most effective of any medicine: for comparison the NNT of statins to prevent the recurrence of a myocardial infarction is about 20.

Similar efficacy is seen in their secondary indications of the treatment of anxiety disorders. Moreover, the desire of many countries to reduce the prescribing of benzodiazepines has lead to a switch to the new antidepressants, particularly the selective serotonin reuptake inhibitors (SSRIs) that have greater efficacy and are much freer from problems such as abuse and withdrawal [4]. Although the SSRIs take several weeks to work and can even worsen anxiety at the start of treatment after a few weeks, they become very effective anxiolytic treatments with efficacy exceeding that of the benzodiazepines. The SSRIs also have uses in other indications such as pain, some sleep disorders and some sexual problems (particularly SSRIs for premature ejaculation).

There are other factors underlying this increase in use. The most important one is that the newer antidepressants are extremely safe drugs. Before their invention, the most commonly used antidepressants were those of tricyclic structure such as amitriptyline and duselepin. However, these are very toxic in overdose due to their combination of noradrenaline reuptake blocking properties and marked anticholinergic actions on the heart. At the peak of their use, they were the most common cause of drug overdose death in the UK, and still today kill hundreds of people a year [5]. Patients with depression are at very high risk of suicide, and before the onset of the SSRIs, many used their antidepressants to kill themselves.

The newer generation of antidepressants exemplified by the SSRIs, but also including venlafaxine, duloxetine, mirtazapine and agomelatine, are orders of magnitude safer than the tricyclics in overdose and much preferred by psychiatrists for this reason. In practice, it is extremely difficult to kill oneself with an overdose of these medicines alone and the cases of suicidal ideation have been minimal and have not lead to actual suicides. The very enhanced safety and tolerability profile of these newer antidepressant drugs has lead to a greater use of them in the depressed population because patients’ compliance with medication is enhanced and doctors are more comfortable prescribing them. This in turn has resulted in a reduction in suicide rate in many countries where their use has become widespread [6].

Despite this, depression is still under-treated with up to half of all patients not being appropriately diagnosed or treated [7]. Efforts to improve this situation are underway, and are more necessary today than was the case a few years ago because depression and suicide rates in Europe are on the rise as a result of the economic downturn [8]. It therefore appears to be the case that the use of antidepressants is large and growing but still inadequate to meet the needs of the population.

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References

1. Nutt DJ: The unhappy saga of ‘happy pills’. J Psychopharmacol 2003, 17:251-252.
2. Murray CJ, Lopez AD: Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet 1997, 349:1498-1504.
3. Geddes JR, Carney SM, Davies C, Furukawa TK, Kupfer DJ, Frank E, Goodwin G: Relapse prevention with antidepressant drug treatment in depressive disorders: a systematic review. Lancet 2003, 361:653-661.
4. Nutt DJ: Death and dependence: current controversies over the selective serotonin reuptake inhibitors. J Psychopharmacol 2003, 17:355-364.
5. Nutt DJ: Death by tricyclic: the real antidepressant scandal? J Psychopharmacol 2005, 19(2):123-125.
6. Isacsson G, Holmgren A, Osby U, Ahlner J: Decrease in suicide among the individuals treated with antidepressants: a controlled study of antidepressants in suicide, Sweden 1995– 2005. Acta Psychiatr Scand 2009, 120(1):37-44.
7. Lecrubier Y: The diagnosis and treatment of depression. CNS Spectr 2008, 13(Suppl. 11):5-9.
8. Stuckler D, Basu S, Suhrcke M, Coutts A, McKee M: Effects of the 2008 recesson on health: a first look at European data. Lancet 2009, 378:124-125.

DOI: 10.1016/j.coph.2011.12.001

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Posted in Pharmacology

Home     Genetics & Development     Pharmacology