Ann Surg Treat Res.  2017 Jul;93(1):11-17. 10.4174/astr.2017.93.1.11.

The effects of indoxyl sulfate-induced endothelial microparticles on neointimal hyperplasia formation in an ex vivo model

• Ryu JH
• Park H
• Kim SJ

• KMID: 2392278
• DOI: http://doi.org/10.4174/astr.2017.93.1.11

Abstract

PURPOSE
Neointimal hyperplasia (NH) is considered to be one of the main causes of vascular access occlusion in patients receiving hemodialysis. Endothelial injury and TGF-β-mediated proliferation of vascular smooth muscle cells (VSMCs) induce NH. Endothelial microparticles (EMPs) are also increased by endothelial injury. We aimed to investigate the effects of EMPs and TGF-β expression on VSMC proliferation and their contributions to NH formation in an ex vivo model.
METHODS
EMPs were collected from the culture media of human umbilical vein endothelial cells treated with indoxyl sulfate (IS, 250 µg/mL) after ultracentrifugation at 100,000 × g. Porcine internal jugular veins were isolated and treated with EMPs (2 × 10⁶ /mL) or left untreated for 12 days and subsequently compared with TGF-β (10 ng/mL)-treated venous tissue. Intima-media thickness and NH area were assessed using a digital program. Masson's trichrome staining and immunohistochemistry (IHC) analysis for α-smooth muscle actin, phosphorylated Akt, ERK1/2, p38 mitogen-activated protein kinase (MAPK), and Smad3 were performed on each vein sample.
RESULTS
NH and VSMC proliferation developed to a significantly greater degree in EMP-treated veins compared to controls, with similar patterns seen in TGF-β-stimulated samples. IHC analysis demonstrated that EMPs markedly increased phosphorylation of Akt, ERK1/2, p38 MAPK, and Smad3 in areas of venous NH formation.
CONCLUSION
Our results showed that IS-induced EMPs provoked massive VSMC proliferation and NH formation via activation of the TGF-β signaling pathways. Further investigation is needed to elucidate the precise mechanism of EMP activity on vascular access stenosis in vivo.

Keyword

Neointima; Transforming Growth factor beta; Cell-derived microparticles

Figure

• 

  Fig. 1 Ex vivo vein model of endothelial microparticles (EMPs) or TGF-β induction of neointimal hyperplasia. Each vessel segment cut into 2-cm pieces was fixed onto a nylon mesh with sterile pins with care to maintain their unfolded morphology, placed in a 100-mm culture dish, covered with 30-mL culture media, and cultured for 12 days. Vessel segments were incubated in 3 different culture conditions: 30% Dulbecco's modified eagle medium (DMEM) media only, EMPs (2 × 106/mL) + DMEM media, and TGF-β (10 ng/mL) + DMEM media. After 12 days, the vessels were preserved in 10% buffered formaldehyde solution until analysis and subsequently compared to uncultured vessel segments. H&E, hematoxylin-eosin; α-SMA, α-smooth muscle actin.

• 

  Fig. 2 Morphological analysis of ex vivo endothelial microparticle (EMP)- and TGF-β-stimulated vein. (A) Representative images of H&E (top row), Masson's trichrome (middle row), and α-smooth muscle actin (SMA) stained (bottom row) ex vivo vein (original magnification, ×100). Evaluation with one-way analysis of variance showed a significantly increased intimal area in the EMP- and TGF-β-treated groups (B) and a prominently increased intimal-medial thickness in EMP- and TGF-β-treated veins (n = 6 in each group) (C). *P < 0.05 vs. D0, ***P < 0.001 vs. D0.

• 

  Fig. 3 Immunohistochemistry assay for TGF-β signaling molecules. Porcine veins after 12 days of endothelial microparticle (EMP) stimulation showed significant (A) neointimal hyperplasia (H&E, ×100) and revealed significantly increased expression of (B) TGF-β and phosphorylation of (C) Akt, (D) ERK1/2, (E) Smad3, and (F) p38 compared to TGF-β-stimulated veins (original magnification, ×100).

Reference

1. Roy-Chaudhury P, Sukhatme VP, Cheung AK. Hemodialysis vascular access dysfunction: a cellular and molecular viewpoint. J Am Soc Nephrol. 2006; 17:1112–1127.

2. Rothuizen TC, Wong C, Quax PH, van Zonneveld AJ, Rabelink TJ, Rotmans JI. Arteriovenous access failure: more than just intimal hyperplasia? Nephrol Dial Transplant. 2013; 28:1085–1092.

3. Heine GH, Ulrich C, Kohler H, Girndt M. Is AV fistula patency associated with angiotensin-converting enzyme (ACE) polymorphism and ACE inhibitor intake? Am J Nephrol. 2004; 24:461–468.

4. Allon M, Robbin ML. Increasing arteriovenous fistulas in hemodialysis patients: problems and solutions. Kidney Int. 2002; 62:1109–1124.

5. Jiang Z, Tao M, Omalley KA, Wang D, Ozaki CK, Berceli SA. Established neointimal hyperplasia in vein grafts expands via TGF-beta-mediated progressive fibrosis. Am J Physiol Heart Circ Physiol. 2009; 297:H1200–H1207.

6. Newby AC, Zaltsman AB. Molecular mechanisms in intimal hyperplasia. J Pathol. 2000; 190:300–309.

7. Tsai S, Hollenbeck ST, Ryer EJ, Edlin R, Yamanouchi D, Kundi R, et al. TGF-beta through Smad3 signaling stimulates vascular smooth muscle cell proliferation and neointimal formation. Am J Physiol Heart Circ Physiol. 2009; 297:H540–H549.

8. Ruiz-Ortega M, Rodriguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J. TGF-beta signaling in vascular fibrosis. Cardiovasc Res. 2007; 74:196–206.

9. Rosenbloom J, Castro SV, Jimenez SA. Narrative review: fibrotic diseases: cellular and molecular mechanisms and novel therapies. Ann Intern Med. 2010; 152:159–166.

10. Kanzaki T, Tamura K, Takahashi K, Saito Y, Akikusa B, Oohashi H, et al. In vivo effect of TGF- beta 1. Enhanced intimal thickening by administration of TGF- beta 1 in rabbit arteries injured with a balloon catheter. Arterioscler Thromb Vasc Biol. 1995; 15:1951–1957.

11. Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transforming growth factor-beta 1 suppress intimal hyperplasia in a rat model. J Clin Invest. 1994; 93:1172–1178.

12. Zegarska J, Paczek L, Pawlowska M, Wyczalkowska A, Michalska W, Ziolkowski J, et al. Increased mRNA expression of transforming growth factor beta in the arterial wall of chronically rejected renal allografts in humans. Transplant Proc. 2006; 38:115–118.

13. Ryan ST, Koteliansky VE, Gotwals PJ, Lindner V. Transforming growth factorbeta-dependent events in vascular remodeling following arterial injury. J Vasc Res. 2003; 40:37–46.

14. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 2009; 19:43–51.

15. Martinez MC, Tesse A, Zobairi F, Andriantsitohaina R. Shed membrane microparticles from circulating and vascular cells in regulating vascular function. Am J Physiol Heart Circ Physiol. 2005; 288:H1004–H1009.

16. Combes V, Simon AC, Grau GE, Arnoux D, Camoin L, Sabatier F, et al. In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest. 1999; 104:93–102.

17. Amabile N, Guerin AP, Leroyer A, Mallat Z, Nguyen C, Boddaert J, et al. Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol. 2005; 16:3381–3388.

18. Ryu JH, Lim SY, Ryu DR, Kang DH, Choi KB, Kim SJ. Association between vascular access failure and microparticles in hemodialysis patients. Kidney Res Clin Pract. 2012; 31:38–47.

19. Jiang Z, Yu P, Tao M, Fernandez C, Ifantides C, Moloye O, et al. TGF-beta- and CTGF-mediated fibroblast recruitment influences early outward vein graft remodeling. Am J Physiol Heart Circ Physiol. 2007; 293:H482–H488.

20. Heaton NS, Wolff RA, Malinowski RL, Hullett DA, Hoch JR. Antisense to transforming growth factor-beta(1) facilitates the apoptosis of macrophages in rat vein grafts. J Vasc Res. 2008; 45:365–374.

21. Khan R, Agrotis A, Bobik A. Understanding the role of transforming growth factor-beta1 in intimal thickening after vascular injury. Cardiovasc Res. 2007; 74:223–234.

22. Wu J, Zhang C. Neointimal hyperplasia, vein graft remodeling, and long-term patency. Am J Physiol Heart Circ Physiol. 2009; 297:H1194–H1195.

23. Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood. 2005; 106:1604–1611.

24. Muller I, Klocke A, Alex M, Kotzsch M, Luther T, Morgenstern E, et al. Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets. FASEB J. 2003; 17:476–478.

25. Mack M, Kleinschmidt A, Bruhl H, Klier C, Nelson PJ, Cihak J, et al. Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection. Nat Med. 2000; 6:769–775.

26. Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, et al. Detection of microRNA expression in human peripheral blood microvesicles. PLoS One. 2008; 3:e3694.

27. Zhang Y, Li L, Yu J, Zhu D, Zhang Y, Li X, et al. Microvesicle-mediated delivery of transforming growth factor β1 siRNA for the suppression of tumor growth in mice. Biomaterials. 2014; 35:4390–4400.

28. Seay U, Sedding D, Krick S, Hecker M, Seeger W, Eickelberg O. Transforming growth factor-beta-dependent growth inhibition in primary vascular smooth muscle cells is p38-dependent. J Pharmacol Exp Ther. 2005; 315:1005–1012.

29. Miyazono K, ten Dijke P, Heldin CH. TGFbeta signaling by Smad proteins. Adv Immunol. 2000; 75:115–157.

30. Eickelberg O. Endless healing: TGF-beta, SMADs, and fibrosis. FEBS Lett. 2001; 506:11–14.