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Questa è la versione dell’autore dell’opera:
Effects Of The Biomimetic Peptide Sh-Polipeptide 9 (CG-VEGF) On
Cocultures Of Human Hair Follicle Dermal Papilla Cells And
Microvascular Endothelial Cells.
Bassino E, Zanardi A, Gasparri F, Munaron L.
Exp Dermatol. 2015 Dec 14. doi: 10.1111/exd.12906
The definitive version is available at:
La versione definitiva è disponibile alla URL:
http://www.ncbi.nlm.nih.gov/pubmed/26662923
Effects Of The Biomimetic Peptide Sh-Polipeptide 9 (CG-VEGF) On Cocultures Of Human Hair Follicle Dermal Papilla Cells And Microvascular Endothelial Cells.
E. Bassino1, A. Zanardi2, F. Gasparri3 & L. Munaron1
1Deparment of Life Sciences and System Biology, University of Turin, Italy. 2 Rottapharm-Madaus, Monza, Italy. 3Department of Pharmacy, University of Salerno, Italy.
Key words
Coculture, endothelial cells, follicle dermal papilla cells, hair growth, androgenetic alopecia. Corresponding author:
Correspondence to: Luca Munaron, Ph.D.
Dept. Life Sciences & Systems Biology University of Torino
Via Accademia Albertina 13 10123 Torino
ITALY
BACKGROUND
Hair follicle (HF) growth require interactions between epithelial and mesenchymal compo-nents and is regulated by angiogenic events driven by microvascular endothelium through the contribution of Vascular Endothelial Growth Factor (VEGF) 1,2,3. Several in vitro and in vivo techniques have been developed to promote hair reconstitution, but further studies are re-quired for their clinical application in regenerative medicine and in particular for Androge-netic Alopecia (AGA) 4. The only approved topical medication for AGA to date is Minoxidil (MXD) 5, that enhances dermal papilla vascularization via VEGF expression and plays a physiological role in maintaining a suitable HF blood supply 6. Nonetheless, there is an in-creasing interest in more powerful and skin absorbable compounds for topic application. In
vitro coculture systems can be successfully employed to test new promising drugs and we
re-cently established transwell-based cocultures to show that follicle dermal papilla cells (FDPC) promote survival, proliferation and tubulogenesis of human microvascular endothe-lial cells (HMVEC) 7. Here we took advantage of this approach to compare the effects in-duced by MXD and Sh-Polypeptide 9, a VEGF-like synthetic biomimetic peptide capsulated by double-layered capsulation technology for better skin penetration. This strategy enhances the transdermal delivery efficacy, stability and purity of the active biomolecule.
QUESTIONS ADDRESSED
The present study investigates the effects of Sh-Polypeptide 9, a VEGF-like biomimetic peptide, on paracrine interactions between endothelial cells and hair follicle papilla dermal cells. These data provide a mechanistic background for its potential use as a promoter of hair follicle vascularization.
EXPERIMENTAL DESIGN See Supplementary material.
RESULTS
Effects of MXD and Sh-Polypeptide 9 on tubulogenesis by HMVEC in monoculture or cocultured with FDPC
Both MXD and Sh-polypeptide 9 were able to enhance HMVEC tubulogenic potential measured by endothelial tube formation assay both in terms of total tubule length and number of junctions (Fig. 1a-c). Interestingly, the presence of FDPC synergized with Sh-Polypeptide-9-induced, but not with MXD-induced tubulogenesis (Fig. 1b, c; see also photographs in Supplementary Fig. 2).
It was previously shown that MXD enhances VEGF production by FDPC 8, but no evidence is available on microvascular endothelium. In nice agreement with the results on tubulogenesis, both the compounds triggered the release of VEGF by starved HMVEC (DMEM 0% FCS; Supplementary Fig. 3 a-c). However, lower concentrations of Sh-Polypeptide 9 resulted significantly more powerful than MXD, being able to increase VEGF levels even more than DMEM 10% FCS (Supplementary Fig. 3; Supplementary Table 1). Sh-Polypeptide 9 stimulated also bFGF release from HMVEC, while MXD induced smaller and non significative effects (Supplementary Fig. 3 d-f).
No additional VEGF or bFGF accumulation was detectable in HMVEC co-cultured with FDPC (Supplementary Fig. 3; Supplementary Table 1).
Effects of MXD and Sh-Polypeptide 9 on viability and proliferation of mono-cultured and co-cultured HMEC and FDPC.
The maximal HMVEC viability was measured when the cells were grown in DMEM 10% FCS (see Methods and Supplementary Fig. 1a for setup configurations) and resulted drastically reduced upon serum deprivation (DMEM 0% FCS, 24 hrs, data not shown).
MXD failed to alter the viability and proliferation of HMVEC as well as of FDPC mono-cultured or co-mono-cultured (Supplementary Fig. 1a, b, e, f, Supplementary Table 2). Differently,
low concentrations of Sh-Polypeptide 9 slightly but significantly increased cell survival of FDPC grown alone: the co-culture with HMVEC enhanced this response (Supplementary Fig. 1c). In addition, the lowest concentration of Sh-Polypeptide 9 was trophic for HMVEC only in the presence of FDPC (Supplementary Fig. 1d).
The lowest concentration of Sh-Polypeptide 9 induced proliferation on both HMVEC and FDPC cultured separately, while no further enhancement was detectable in co-cultures (Supplementary Fig. 1g, h; Supplementary Table 2).
Effects of MXD and Sh-Polypeptide 9 on -catenin nuclear expression of FDPC in monoculture or cocultured with HMVEC.
In FDPC cultured alone, the lowest concentrations of MXD (Fig. 2 b) and Sh-Polypeptide 9 (Fig. 2c) stimulated the production of -catenin, an intracellular pleiotropic signaling molecule involved in the regulation of follicle cell adhesion and signaling. The effect of Sh-Polypeptide 9 was significantly more pronounced and coculture with HMVEC did not significantly interfere on the response (Fig. 2b, c; Supplementary Table 1). These evidences confirm the previous observations suggesting that the lower concentrations of the drugs are more effective.
Effects of MXD and Sh-Polypeptide 9 on Interleukin-1 (IL-1and caspase-3 expression by FDPC in monoculture or cocultured with HMVEC.
Finally, we asked whether the two drugs could protect endothelium against oxidative stress occurring during aging, skin wounding and inflammatory responses. Since IL-1 is a cytokine prominent in these events, and acts as a negative hair growth regulator, we decided to evaluate the effects of MXD and Sh-Polypeptide 9 on its endothelial release. HMVEC were treated with H2O2 (400 M, 2 hrs) to induce a strong oxidative stress. As expected, this stimulation evoked a strong increase of IL-1levels in endothelial medium (24 hrs). The lowest MXD concentration slightly (but non significantly) reduced IL-1 production, that
was dramatically prevented by the lowest concentration of Sh-Polypeptide 9 (Fig. 2e, Supplementary Table 1). The same trend was measured for the apoptotic marker caspase-3 (Fig. 2f). No further changes were observed in the presence of FDPC.
CONCLUSIONS
We have recently shown that FDPC strongly support endothelial survival, proliferation and tubulogenesis in insert-based co-cultures 7.
Here this approach was employed to investigate the activity of MXD and Sh-Polypeptide 9 (VEGF-like biomimetic peptide) on HMEC-FDPC cross-talk.
The results reveal that Sh-Polypeptide 9 promotes endothelial tubulogenesis and VEGF production more efficiently than MXD, in particular at low concentrations. In addition, the VEGF-like biomimetic peptide is more powerful on -catenin expression by FDPC and on endothelial protection from oxidative stress.
In conclusion, our in vitro study points to Sh-Polypeptide 9 as a promising molecule: however, only further detailed mechanistic studies, together with ‘in vivo’ and clinical trials, will establish its positive effects against hair loss.
Acknowledgements
E.B. performed the research, analyzed the data and wrote the paper F.G. designed the research study
A.Z. contributed essential reagents
L.M. designed the research study, wrote the paper and contributed essential reagents. We acknowledge funding from Rottapharm-Madaus.
Conflicts of interest: None declared REFERENCES
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FIGURE LEGENDS Figure 1.
Tubulogenic activity of MXD or Sh-Polypeptide 9. a) Experimental configurations of co-cultures (A-D).
b,c) Tubulogenesis at 18 hrs in mono- or co-cultured HMVEC grown in EGM-2 or DMEM 10% medium and in the presence of MXD or Sh-Polypeptide 9.
Figure 2.
Protective activity of MXD or Sh-Polypeptide 9. a) Experimental configurations of co-cultures (A-D).
b-c) -catenin expression by mono- or cocultured FDPC in the presence of MDX or Sh-Polypeptide 9.
MXD (0. 2 mg/ml (1), 0.1 mg/ml (2), 0.01 mg/ml (3))
Sh-Polypeptide 9 (10 mg/ml (1), 5 mg/ml (2), 2.5 mg/ml (3)) d) Experimental configurations of co-cultures (E-F).
e,f) Evaluation of L- and caspase-3 expression on mono- or co-cultured HMVEC treated with H2O2 and in the presence of MXD or Sh-Polypeptide 9.
MXD (0.01 mg/ml (3))
Sh-Polypeptide 9 (2.5 mg/ml (3))
