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Research Note

Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation

[version 1; peer review: 2 approved, 1 approved with reservations]
PUBLISHED 27 Jan 2015
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Abstract

Accelerated vascular smooth muscle cell (VSMC) proliferation is implied in cardiovascular disease and significantly contributes to vessel lumen reduction following surgical interventions such as percutaneous transluminal coronary angioplasty or bypass surgery. Therefore, identification and characterization of compounds and mechanisms able to counteract VSMC proliferation is of potential therapeutic relevance. This work reveals the anti-proliferative effect of the natural product capsaicin from Capsicum spp. by quantification of metabolic activity and DNA synthesis in activated VSMC. The observed in vitro activity profile of capsaicin warrants further research on its mechanism of action and potential for therapeutic application.

Keywords

Capsaicin, vascular smooth muscle cells, restenosis, proliferation

Main text

Aberrant and accelerated VSMC proliferation is a main contributor to restenosis, the pathological re-narrowing of the vessel lumen after surgical interventions combating vascular stenosis. To overcome restenosis, drug-eluting stents have been developed, aiming at inhibiting VSMC growth by the release of anti-proliferative substances such as paclitaxel and rapamycin. However, these compounds display unresolved issues such as impaired re-endothelialization and subsequent thrombosis induction1, which makes the characterization of other compounds able to suppress VSMC proliferation highly relevant. Plant-derived natural products are an excellent resource for identifying lead compounds2. Here we examine the anti-proliferative potential of capsaicin, a bioactive component of chili peppers [Capsicum spp. (Solanaceae)], in VSMC.

To test whether capsaicin is able to inhibit proliferation of VSMC induced by PDGF, a major growth factor implied in the aberrant proliferative responses in restenosis3, the total amount of metabolically active cells was measured after 48 h of incubation by the resazurin conversion method4. Capsaicin indeed suppressed VSMC proliferation concentration-dependently with an IC50 of 5.36 μM (Figure 1A). To confirm the anti-proliferative effect of capsaicin with a second experimental method, we measured DNA synthesis in VSMC by quantification of 5-bromo-2′-deoxyuridine (BrdU) incorporation into DNA. Capsaicin also inhibited PDGF-stimulated DNA synthesis in a concentration-dependent manner with an IC50 of 3.81 μM (Figure 1B). To assure that the decreased number of VSMC upon treatment with capsaicin is not due to cytotoxicity, we quantified cell death by measuring cell membrane integrity estimated by lactate dehydrogenase (LDH) activity inside cells and in cell supernatants. No significant cytotoxicity was detected in the investigated concentration range (Figure 1C). In summary, capsaicin is identified as an inhibitor of VSMC proliferation. Further studies are prompted to elaborate the underlying mode of action of this natural product and to investigate its effect in advanced in vivo anti-restenotic models.

98d2f0aa-b2fa-483f-822e-8efc2c0206d8_figure1.gif

Figure 1. Effect of capsaicin on VSMC proliferation.

Cell proliferation was estimated by quantification of metabolic activity (A) and DNA synthesis (B). Cell death was estimated by quantification of the percentage of extracellular LDH (C). Data represent mean ± SD from at least three independent experiments (n.s., not significant; ***p < 0.001; **p < 0.01; ANOVA/Bonferroni).

Rat aortic VSMC used in this study were purchased from Lonza (Braine-L’Alleud, Belgium) and cultivated in DMEM–F12 (1:1) medium supplemented with 20% fetal calf serum and gentamycin. Capsaicin and other chemicals were obtained from Sigma-Aldrich (Vienna, Austria).

For the resazurin conversion assay, VSMC were seeded in 96-well plates at 5 × 103 cells/well. 24 h later, cells were serum-starved for 24 h to render them quiescent. Quiescent cells were pretreated for 30 min with capsaicin or vehicle (0.1% DMSO) as indicated, and subsequently stimulated for 48 h with PDGF-BB (20 ng/mL). To measure the number of metabolically active VSMC by resazurin conversion4, cells were washed with PBS and incubated in serum-free medium containing 10 μg/mL resazurin for 2 h. Total metabolic activity was measured by monitoring the increase in fluorescence at a wavelength of 590 nm using an excitation wavelength of 535 nm in a 96-well plate reader (Tecan GENios Pro).

For the BrdU incorporation assay, VSMC were seeded and starved as for the resazurin conversion assay. Quiescent cells were pretreated for 30 min with capsaicin, or vehicle as indicated and subsequently stimulated with PDGF-BB (20 ng/mL). To estimate de novo DNA synthesis in VSMC5, BrdU was added 2 h after PDGF stimulation, and the incorporated amount was determined 22 h afterwards with a BrdU ELISA kit according to the manufacturer’s instructions (Roche Diagnostics).

For assessing cytotoxicity, VSMC were seeded and serum-starved as indicated above. The quiescent cells were pretreated for 30 min with capsaicin, or vehicle as indicated, and subsequently stimulated for 24 h with PDGF-BB (20 ng/mL). To quantify the loss of cell membrane integrity as a sign for cell death6, the supernatants of the treated cells were assessed for LDH activity. For estimation of the total LDH, identically treated samples were incubated for 45 min in the presence of 1% Triton X-100. The released and total LDH enzyme activity was quantified for 30 min in the dark in the presence of 4.5 mg/mL lactate, 0.56 mg/mL NAD+, 1.69 U/mL diaphorase, 0.004% (w/v) BSA, 0.15% (w/v) sucrose, and 0.5 mM 2-p-iodophenyl-3-nitrophenyl tetrazolium chloride (INT). The enzyme reaction was stopped with 1.78 mg/mL oxymate and the absorbance was measured at 490 nm in a 96-well plate reader (Tecan GENios Pro). Potential effects on cell viability were estimated as percentage of extracellular LDH activity. The cytotoxic natural product digitonin (100 μg/mL) was used as a positive control.

Statistical analysis was performed by ANOVA/Bonferroni test (GraphPad PRISM software, version 4).

Data availability

Figshare: Effect of capsaicin on vascular smooth muscle cell proliferation: raw data. doi: 10.6084/m9.figshare.12897217

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Liu R, Heiss EH, Guo D et al. Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation [version 1; peer review: 2 approved, 1 approved with reservations]. F1000Research 2015, 4:26 (https://doi.org/10.12688/f1000research.6007.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 27 Jan 2015
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63
Cite
Reviewer Report 02 Feb 2015
Karel Šmejkal, Department of Natural Drugs, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic 
Approved
VIEWS 63
The authors are describing the inhibitory activity of capsaicin on vascular smooth muscle cell proliferation. The article looks to ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Šmejkal K. Reviewer Report For: Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation [version 1; peer review: 2 approved, 1 approved with reservations]. F1000Research 2015, 4:26 (https://doi.org/10.5256/f1000research.6428.r7485)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 29 Apr 2015
    Atanas Atanasov, Department of Pharmacognosy, University of Vienna, Vienna, Austria
    29 Apr 2015
    Author Response
    Thank you very much for taking the time to review our manuscript.
     
    Aiming to identify new inhibitors of VSMC proliferation, we tested a range of natural compounds derived from medicinal plants ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 29 Apr 2015
    Atanas Atanasov, Department of Pharmacognosy, University of Vienna, Vienna, Austria
    29 Apr 2015
    Author Response
    Thank you very much for taking the time to review our manuscript.
     
    Aiming to identify new inhibitors of VSMC proliferation, we tested a range of natural compounds derived from medicinal plants ... Continue reading
Views
71
Cite
Reviewer Report 02 Feb 2015
Goutam Brahmachari, Laboratory of Natural Products & Organic Synthesis, Department of Chemistry, Visva-Bharati University, Santiniketan, West Bengal, India 
Approved
VIEWS 71
I have gone through the present manuscript entitled “Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation” by Liu et al. with interest where the investigators demonstrated the efficacy of natural capsaicin in overcoming restenosis by using a ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Brahmachari G. Reviewer Report For: Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation [version 1; peer review: 2 approved, 1 approved with reservations]. F1000Research 2015, 4:26 (https://doi.org/10.5256/f1000research.6428.r7484)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 29 Apr 2015
    Atanas Atanasov, Department of Pharmacognosy, University of Vienna, Vienna, Austria
    29 Apr 2015
    Author Response
    Thank you very much for taking the time to review our manuscript.
     
    Based on the literature, as well as on our own data, the both suggested reference compounds, paclitaxel and rapamycin, ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 29 Apr 2015
    Atanas Atanasov, Department of Pharmacognosy, University of Vienna, Vienna, Austria
    29 Apr 2015
    Author Response
    Thank you very much for taking the time to review our manuscript.
     
    Based on the literature, as well as on our own data, the both suggested reference compounds, paclitaxel and rapamycin, ... Continue reading
Views
81
Cite
Reviewer Report 30 Jan 2015
Carsten Gründemann, Center for Complementary Medicine, Universitätsklinikum Freiburg, Freiburg, Germany 
Approved with Reservations
VIEWS 81
In the current manuscript, Rongxia Liu et al investigated the influence of the plant-derived natural product capsaicin to overcome restenosis by using a vascular muscle cell model
(VSCM) in vitro.

The manuscript is well written and conclusion is precise. The experiments are ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Gründemann C. Reviewer Report For: Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation [version 1; peer review: 2 approved, 1 approved with reservations]. F1000Research 2015, 4:26 (https://doi.org/10.5256/f1000research.6428.r7483)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 29 Apr 2015
    Atanas Atanasov, Department of Pharmacognosy, University of Vienna, Vienna, Austria
    29 Apr 2015
    Author Response
    Thank you very much for taking the time to review our manuscript.
     
    While reporting a new bioactivity of the interesting natural product capsaicin, the purpose of our short note is not ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 29 Apr 2015
    Atanas Atanasov, Department of Pharmacognosy, University of Vienna, Vienna, Austria
    29 Apr 2015
    Author Response
    Thank you very much for taking the time to review our manuscript.
     
    While reporting a new bioactivity of the interesting natural product capsaicin, the purpose of our short note is not ... Continue reading

Comments on this article Comments (1)

Version 1
VERSION 1 PUBLISHED 27 Jan 2015
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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