Limits...
Human platelet-rich plasma- and extracellular matrix-derived peptides promote impaired cutaneous wound healing in vivo.

Demidova-Rice TN, Wolf L, Deckenback J, Hamblin MR, Herman IM - PLoS ONE (2012)

Bottom Line: Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma.In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury.Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, The Center for Innovations in Wound Healing Research, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds. Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment. Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.

Show MeSH

Related in: MedlinePlus

Platelet rich plasma derived peptides stimulate in vitro angiogenesis and epithelial responses to injury.A – In vitro angiogenesis assay. Human capillary endothelial cells were plated on growth factor reduced Matrigel as described in Materials and Methods. The media was supplemented with either DMEM supplemented with 1% BCS or 250 nM of UN1, UN2 or UN3 peptides. Cells that have received DMEM/1% in the presence of 10 ng/ml VEGF served as positive control. Total tube length was measured at 4 h post-plating. Relative tube length compared to control is shown. B – In vitro epithelial wound healing assay. NHEK cells were plated and injured as described in Material ad methods. The following experimental conditions were used: basal keratinocyte growth media (control), 10 ng/ml HB-EGF (positive control), 0.5 nM UN1, UN2 or UN3 peptides. Relative wound closure is shown. Data are presented as mean +/− standard error; * - indicates p<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3285658&req=5

pone-0032146-g004: Platelet rich plasma derived peptides stimulate in vitro angiogenesis and epithelial responses to injury.A – In vitro angiogenesis assay. Human capillary endothelial cells were plated on growth factor reduced Matrigel as described in Materials and Methods. The media was supplemented with either DMEM supplemented with 1% BCS or 250 nM of UN1, UN2 or UN3 peptides. Cells that have received DMEM/1% in the presence of 10 ng/ml VEGF served as positive control. Total tube length was measured at 4 h post-plating. Relative tube length compared to control is shown. B – In vitro epithelial wound healing assay. NHEK cells were plated and injured as described in Material ad methods. The following experimental conditions were used: basal keratinocyte growth media (control), 10 ng/ml HB-EGF (positive control), 0.5 nM UN1, UN2 or UN3 peptides. Relative wound closure is shown. Data are presented as mean +/− standard error; * - indicates p<0.05.

Mentions: As shown in Figure 3A, fractionation of platelet extracts significantly reduced the complexity of the protein mixtures and eliminated all high molecular weight proteins. Mass spectrometry analysis of the protein bands with molecular weight below 150 kd revealed several unnamed (UN) protein fragments, some of which possess sequence identity to fragments of human thrombin. Corresponding peptides were synthesized at TUCF. The peptides sequences are as follows: NH2-ELLESYIDGR-Amide – UN1; NH2- TATSEYQTFFNPR-Amide – UN2; NH2-ELLESYIDGRPTATSEYQTFFNPR-Amide – UN3. All three peptides stimulated angiogenesis in vitro as well as epithelial migration. However, as shown in Figure 4 a combinatorial UN3 peptide was a more potent stimulator of cellular responses than either UN1 or UN2 peptides. It induced 2.5-fold increase in a total tube length and a 3-fold increase in epithelial responses to injury (Figure 4 A and B). Therefore, we chose to use this peptide for our further in vitro and in vivo experiments.


Human platelet-rich plasma- and extracellular matrix-derived peptides promote impaired cutaneous wound healing in vivo.

Demidova-Rice TN, Wolf L, Deckenback J, Hamblin MR, Herman IM - PLoS ONE (2012)

Platelet rich plasma derived peptides stimulate in vitro angiogenesis and epithelial responses to injury.A – In vitro angiogenesis assay. Human capillary endothelial cells were plated on growth factor reduced Matrigel as described in Materials and Methods. The media was supplemented with either DMEM supplemented with 1% BCS or 250 nM of UN1, UN2 or UN3 peptides. Cells that have received DMEM/1% in the presence of 10 ng/ml VEGF served as positive control. Total tube length was measured at 4 h post-plating. Relative tube length compared to control is shown. B – In vitro epithelial wound healing assay. NHEK cells were plated and injured as described in Material ad methods. The following experimental conditions were used: basal keratinocyte growth media (control), 10 ng/ml HB-EGF (positive control), 0.5 nM UN1, UN2 or UN3 peptides. Relative wound closure is shown. Data are presented as mean +/− standard error; * - indicates p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285658&req=5

pone-0032146-g004: Platelet rich plasma derived peptides stimulate in vitro angiogenesis and epithelial responses to injury.A – In vitro angiogenesis assay. Human capillary endothelial cells were plated on growth factor reduced Matrigel as described in Materials and Methods. The media was supplemented with either DMEM supplemented with 1% BCS or 250 nM of UN1, UN2 or UN3 peptides. Cells that have received DMEM/1% in the presence of 10 ng/ml VEGF served as positive control. Total tube length was measured at 4 h post-plating. Relative tube length compared to control is shown. B – In vitro epithelial wound healing assay. NHEK cells were plated and injured as described in Material ad methods. The following experimental conditions were used: basal keratinocyte growth media (control), 10 ng/ml HB-EGF (positive control), 0.5 nM UN1, UN2 or UN3 peptides. Relative wound closure is shown. Data are presented as mean +/− standard error; * - indicates p<0.05.
Mentions: As shown in Figure 3A, fractionation of platelet extracts significantly reduced the complexity of the protein mixtures and eliminated all high molecular weight proteins. Mass spectrometry analysis of the protein bands with molecular weight below 150 kd revealed several unnamed (UN) protein fragments, some of which possess sequence identity to fragments of human thrombin. Corresponding peptides were synthesized at TUCF. The peptides sequences are as follows: NH2-ELLESYIDGR-Amide – UN1; NH2- TATSEYQTFFNPR-Amide – UN2; NH2-ELLESYIDGRPTATSEYQTFFNPR-Amide – UN3. All three peptides stimulated angiogenesis in vitro as well as epithelial migration. However, as shown in Figure 4 a combinatorial UN3 peptide was a more potent stimulator of cellular responses than either UN1 or UN2 peptides. It induced 2.5-fold increase in a total tube length and a 3-fold increase in epithelial responses to injury (Figure 4 A and B). Therefore, we chose to use this peptide for our further in vitro and in vivo experiments.

Bottom Line: Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma.In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury.Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, The Center for Innovations in Wound Healing Research, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds. Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment. Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.

Show MeSH
Related in: MedlinePlus