World J Mens Health.  2020 Jan;38(1):123-131. 10.5534/wjmh.180091.

A Simple and Nonenzymatic Method to Isolate Human Corpus Cavernosum Endothelial Cells and Pericytes for the Study of Erectile Dysfunction

• Yin GN
• Ock J
• Choi MJ
• Song KM
• Ghatak K
• Minh NN
• Kwon MH
• Seong DH
• Jin HR
• Ryu JK
• Suh JK

• KMID: 2465417
• DOI: http://doi.org/10.5534/wjmh.180091

Abstract

PURPOSE
To establish a simple and nonenzymatic technique to isolate endothelial cells (ECs) and pericytes from human corpus cavernosum tissue and to evaluate the angiogenic ability of the human cavernous EC or pericytes for the study of high glucose-induced angiopathy.
MATERIALS AND METHODS
For primary human cavernous EC culture, cavernous tissues were implanted into Matrigel in dishes. For primary human cavernous pericyte culture, cavernous tissues were settled by gravity into dishes. We performed immunocytochemistry and Western blot to determine phenotype and morphologic changes from passage 1 to 5. The primary cultured cells were exposed to a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition, and then tube formation assay was done.
RESULTS
We successfully isolated high-purity EC and pericytes from human corpus cavernosum tissue. Primary cultured EC showed highly positive staining for von Willebrand factor, and pericyte revealed positive staining for NG2 and platelet-derived growth factor receptor-β. Primary cultured EC and pericytes maintained their cellular characteristics up to passage 2 or 3. However, we observed significant changes in their typical phenotype from the passage 4 and morphological characteristics from the passage 3. Human cavernous EC or pericytes formed well-organized capillary-like structures in normal-glucose condition, whereas severely impaired tube formation was detected in high-glucose condition.
CONCLUSIONS
This study provides a simple and nonenzymatic method for primary culture of human cavernous EC and pericytes. Our study will aid us to understand the pathophysiology of diabetic erectile dysfunction, and also be a valuable tool for determining the efficacy of candidate therapeutic targets.

Keyword

Diabetes mellitus; Endothelial cell; Erectile dysfunction; Pericytes

MeSH Terms

Blotting, Western
Cells, Cultured
Diabetes Mellitus
Endothelial Cells*
Erectile Dysfunction*
Gravitation
Humans*
Immunohistochemistry
Male
Methods*
Pericytes*
Phenotype
Platelet-Derived Growth Factor
von Willebrand Factor
Platelet-Derived Growth Factor
von Willebrand Factor

Figure

• 

  Fig. 1 Localization of endothelial cells and pericytes in human corpus cavernosum tissue. Immunofluorescent staining of human penile tissue (n=3) performed with antibodies against CD34 or von Willebrand factor (vWF, endothelial cell markers, red) and platelet-derived growth factor receptor-β (PDGFR-β) or NG2 (pericyte markers, green). Scale bar=25 µm. DAPI: 4,6-diamidino-2-phenylindole (a nuclei marker, blue).

• 

  Fig. 2 Isolation and characterization of human cavernous endothelial cells and pericytes. (A) The human corpus cavernosum tissues were implanted on a Matrigel-coated 60-mm cell culture dish with endothelial cell culture medium. A representative image of the cells cultivated for 14 days. After cells were confluent and spread over the whole bottom (21 days), only sprouting cells were used for subcultivation. (B) Fluorescent immunocytochemistry of primary human cavernous endothelial cells (passage 1) with antibodies against von Willebrand factor (vWF, endothelial cell marker) and NG2 (a pericyte marker). Nuclei were labeled with the DNA dye DAPI (4,6-diamidino-2-phenylindole). Scale bar=100 µm. (C) The human corpus cavernosum tissues were implanted on a collagen I-coated 35-mm cell culture dishes with pericyte culture medium. A representative image of the cells cultivated for 14 days. After cells were confluent and spread over the whole bottom (21 days), only sprouting cells were used for subcultivation. (D) Fluorescent immunocytochemistry of primary human cavernous pericytes (passage 1) with antibodies against vWF and NG2. Scale bar=100 µm.

• 

  Fig. 3 Morphologic and phenotypic changes of human cavernous endothelial cells (ECs) according to the passages (Ps). (A) Phase image of human cavernous ECs from P1 to P5. (B) Fluorescent immunocytochemistry of human cavernous ECs with antibody against CD34 or von Willebrand factor (vWF, EC markers) and antibody against platelet-derived growth factor receptor-β (PDGFR-β) or NG2 (pericyte markers). Nuclei were labeled with the DNA dye DAPI (4,6-diamidino-2-phenylindole). Scale bar=100 µm. (C) The percentage of CD34- or vWF-positive cells was quantified by Image J. *p<0.05 compared with P1 to P3 groups. (D) Representative Western blot for CD34. (E) Data are presented as the relative density of CD34 to β-actin. The relative ratio measured in the P1 group is arbitrarily presented as 1. *p<0.05 compared with P1 group. **p<0.01 compared with P2 to P3 group. Each bar depicts the mean values (±standard error) from four experiments per group.

• 

  Fig. 4 Morphologic and phenotypic changes of human cavernous pericytes according to the passages (Ps). (A) Phase image of human cavernous pericytes from P1 to P5. (B) Fluorescent immunocytochemistry of human cavernous pericytes with antibody against platelet-derived growth factor receptor-β (PDGFR-β) or NG2 (pericyte markers) and antibody against CD34 or von Willebrand factor (vWF, endothelial cell markers). Nuclei were labeled with the DNA dye DAPI (4,6-diamidino-2-phenylindole). Scale bar=100 µm. (C) The percentage of PDGFR-β or NG2-positive pericytes was quantified by Image J. *p<0.05 compared with P1 to P3 groups. (D) Representative Western blot for PDGFR-β. (E) Data are presented as the relative density of PDGFR-β to β-actin. The relative ratio measured in the P1 group is arbitrarily presented as 1. *p<0.05 compared with P1 to P2 groups. **p<0.01 compared with P3 groups. Each bar depicts the mean values (±standard error) from four experiments per group.

• 

  Fig. 5 Differentiation of human cavernous endothelial cells and pericytes into fibroblast-like cells at passage (P) 5. (A, B) Fluorescent immunocytochemistry of human cavernous endothelial cells with antibody against fibroblast-specific protein 1 (FSP1, a fibroblast marker) or von Willebrand factor (vWF, an endothelial cell marker) and antibody against FSP1 or platelet-derived growth factor receptor-β (PDGFR-β). Nuclei were labeled with the DNA dye DAPI (4,6-diamidino-2-phenylindole). Scale bar=100 µm. (C, D) The percentage of FSP1 positive cells in P1 and P5 were quantified by Image J. ***p<0.001 compared with P1 groups. Each bar depicts the mean values (±standard error) from four experiments per group.

• 

  Fig. 6 Decrease in the number of tubes formed in human cavernous endothelial cells or pericytes exposed to the high-glucose (HG) condition. Phase-contrast microscopy of human cavernous endothelial cells (A) and human cavernous pericytes (B). After serum starvation for 24 hours, human cavernous endothelial cells and pericytes were incubated in the normal-glucose (NG, 5 mM) or the HG (30 mM) condition for 48 hours. Then, the tube formation assay on Matrigel was performed in 96-well dishes (×40 magnification). (C, D) Number of branch points per field. Each bar depicts the mean values (±standard error) from four separate wells per group. **p<0.01 compared with the NG group.

Reference

1. Andersson KE. Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacol Rev. 2011; 63:811–859. PMID: 21880989.

2. Yin GN, Das ND, Choi MJ, Song KM, Kwon MH, Ock J, et al. The pericyte as a cellular regulator of penile erection and a novel therapeutic target for erectile dysfunction. Sci Rep. 2015; 5:10891. PMID: 26044953.

3. Yin GN, Park SH, Choi MJ, Limanjaya A, Ghatak K, Minh NN, et al. Penile neurovascular structure revisited: immunohistochemical studies with three-dimensional reconstruction. Andrology. 2017; 5:964–970. PMID: 28805947.

4. Verma S, Buchanan MR, Anderson TJ. Endothelial function testing as a biomarker of vascular disease. Circulation. 2003; 108:2054–2059. PMID: 14581384.

5. Díaz-Flores L, Gutiérrez R, Varela H, Rancel N, Valladares F. Microvascular pericytes: a review of their morphological and functional characteristics. Histol Histopathol. 1991; 6:269–286. PMID: 1802127.

6. Bauer AL, Jackson TL, Jiang Y. A cell-based model exhibiting branching and anastomosis during tumor-induced angiogenesis. Biophys J. 2007; 92:3105–3121. PMID: 17277180.

7. Bookholt FD, Monsuur HN, Gibbs S, Vermolen FJ. Mathematical modelling of angiogenesis using continuous cell-based models. Biomech Model Mechanobiol. 2016; 15:1577–1600. PMID: 27037954.

8. Finkenzeller G, Torio-Padron N, Momeni A, Mehlhorn AT, Stark GB. In vitro angiogenesis properties of endothelial progenitor cells: a promising tool for vascularization of ex vivo engineered tissues. Tissue Eng. 2007; 13:1413–1420. PMID: 17550338.

9. Baudin B, Bruneel A, Bosselut N, Vaubourdolle M. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc. 2007; 2:481–485. PMID: 17406610.

10. Bryan BA, D'Amore PA. Pericyte isolation and use in endothelial/pericyte coculture models. Methods Enzymol. 2008; 443:315–331. PMID: 18772023.

11. Maier CL, Shepherd BR, Yi T, Pober JS. Explant outgrowth, propagation and characterization of human pericytes. Microcirculation. 2010; 17:367–380. PMID: 20618694.

12. Mogensen C, Bergner B, Wallner S, Ritter A, d'Avis S, Ninichuk V, et al. Isolation and functional characterization of pericytes derived from hamster skeletal muscle. Acta Physiol (Oxf). 2011; 201:413–426. PMID: 20969729.

13. Rops AL, van der Vlag J, Jacobs CW, Dijkman HB, Lensen JF, Wijnhoven TJ, et al. Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines. Kidney Int. 2004; 66:2193–2201. PMID: 15569308.

14. Tigges U, Welser-Alves JV, Boroujerdi A, Milner R. A novel and simple method for culturing pericytes from mouse brain. Microvasc Res. 2012; 84:74–80. PMID: 22484453.

15. Bernas MJ, Cardoso FL, Daley SK, Weinand ME, Campos AR, Ferreira AJG, et al. Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier. Nat Protoc. 2010; 5:1265–1272. PMID: 20595955.

16. Yin GN, Ryu JK, Kwon MH, Shin SH, Jin HR, Song KM, et al. Matrigel-based sprouting endothelial cell culture system from mouse corpus cavernosum is potentially useful for the study of endothelial and erectile dysfunction related to high-glucose exposure. J Sex Med. 2012; 9:1760–1772. PMID: 22548733.

17. Bala K, Ambwani K, Gohil NK. Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: implication on use of higher passage cells. Tissue Cell. 2011; 43:216–222. PMID: 21511321.

18. Neng L, Zhang W, Hassan A, Zemla M, Kachelmeier A, Fridberger A, et al. Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear. Nat Protoc. 2013; 8:709–720. PMID: 23493068.

19. Allen DA, Harwood S, Varagunam M, Raftery MJ, Yaqoob MM. High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases. Faseb J. 2003; 17:908–910. PMID: 12670885.

20. Rajesh M, Mukhopadhyay P, Bátkai S, Haskó G, Liaudet L, Drel VR, et al. Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption. Am J Physiol Heart Circ Physiol. 2007; 293:H610–H619. PMID: 17384130.

21. Yu T, Sheu SS, Robotham JL, Yoon Y. Mitochondrial fission mediates high glucose-induced cell death through elevated production of reactive oxygen species. Cardiovasc Res. 2008; 79:341–351. PMID: 18440987.

22. Langley RR, Ramirez KM, Tsan RZ, Van Arsdall M, Nilsson MB, Fidler IJ. Tissue-specific microvascular endothelial cell lines from H-2K(b)-tsA58 mice for studies of angiogenesis and metastasis. Cancer Res. 2003; 63:2971–2976. PMID: 12782605.

23. Moon KH, Park SY, Kim YW. Obesity and erectile dysfunction: from bench to clinical implication. World J Mens Health. 2019; 37:138–147. PMID: 30079640.

24. Armulik A, Genové G, Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev Cell. 2011; 21:193–215. PMID: 21839917.

25. Armulik A, Abramsson A, Betsholtz C. Endothelial/pericyte interactions. Circ Res. 2005; 97:512–523. PMID: 16166562.

26. Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol. 2007; 9:255–267. PMID: 17293855.

27. Capetandes A, Gerritsen ME. Simplified methods for consistent and selective culture of bovine retinal endothelial cells and pericytes. Invest Ophthalmol Vis Sci. 1990; 31:1738–1744. PMID: 2120144.

28. Helmbold P, Nayak RC, Marsch WC, Herman IM. Isolation and in vitro characterization of human dermal microvascular pericytes. Microvasc Res. 2001; 61:160–165. PMID: 11254395.

29. Yin GN, Park SH, Song KM, Limanjaya A, Ghatak K, Minh NN, et al. Establishment of in vitro model of erectile dysfunction for the study of high-glucose-induced angiopathy and neuropathy. Andrology. 2017; 5:327–335. PMID: 27992968.