140 Chapter 6 Stress Removal and In Vitro Wound Healing Activity of Peptides C2 and E1 6.1 INTRODUCTION ROS are required by living systems to maintain a number of functions including signal transduction, cell adhesion and wound healing (Park et al 2011). When the delicate balance between the ROS creation and depletion is hampered due to excess ROS or faulty detoxification mechanism, oxidative stress ensues. Excess ROS acts through multiple mechanisms to cause stress: oxidizes cellular macromolecules leading to membrane damage, enzyme dysfunction and metabolic flux hamper and impaired DNA repair, resulting in mutagenesis or cell death (Lum and Roebuck, 2001). During wound repair, reactive radicals created by neutrophils in the second phase of wound healing serves as signals for other group of cells to initiate steps for the third and fourth phase of wound healing. However, in case of persistent wounds or the presence of foreign elements near a wound site, the resultant uncontrolled stress, ROS or otherwise, can lead to deleterious effects. Although very low levels of free radicals can actually increase the integrin attachment to ECM, the effect is reversed at higher levels. Excess ROS displays a multitude of effects including altered integrin sub-unit gene expression, consequently leading to improper connectivity with the ECM and eventually, cell death (Lamari et al 2007; Mian et al 2008). Oxidative stress also results in reorganization of actin filaments through FAK, resulting in loss of cell-matrix adhesiveness. This may eventually lead to cell loss, compromised tissue integrity, activation of apoptotic pathways and pathologic consequences (Zhou et al 1999). It is hypothesized that this cell loss in the presence of ROS stress can be countered by an efficient cell adhesion. Thus, components of the matrix with an increased cell adhesive ability could provide some form of protection to stress-exposed cells. The increased cell survival, in turn can also have a substantial effect on cell movement during in vivo stress-generating events like tissue remodelling and wound closure. The physiological effects of oxidative stress in vitro can be mimicked by the use of exogenously administered H 2 O 2 and by heavy metals. ROS stress can be generated by metals through two distinct mechanisms: With redox-active metals like Fe and Cr, a
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140
Chapter 6
Stress Removal and In Vitro Wound Healing Activity of
Peptides C2 and E1
6.1 INTRODUCTION
ROS are required by living systems to maintain a number of functions including
signal transduction, cell adhesion and wound healing (Park et al 2011). When the delicate
balance between the ROS creation and depletion is hampered due to excess ROS or faulty
detoxification mechanism, oxidative stress ensues. Excess ROS acts through multiple
mechanisms to cause stress: oxidizes cellular macromolecules leading to membrane
damage, enzyme dysfunction and metabolic flux hamper and impaired DNA repair,
resulting in mutagenesis or cell death (Lum and Roebuck, 2001).
During wound repair, reactive radicals created by neutrophils in the second phase
of wound healing serves as signals for other group of cells to initiate steps for the third
and fourth phase of wound healing. However, in case of persistent wounds or the
presence of foreign elements near a wound site, the resultant uncontrolled stress, ROS or
otherwise, can lead to deleterious effects. Although very low levels of free radicals can
actually increase the integrin attachment to ECM, the effect is reversed at higher levels.
Excess ROS displays a multitude of effects including altered integrin sub-unit gene
expression, consequently leading to improper connectivity with the ECM and eventually,
cell death (Lamari et al 2007; Mian et al 2008). Oxidative stress also results in
reorganization of actin filaments through FAK, resulting in loss of cell-matrix
adhesiveness. This may eventually lead to cell loss, compromised tissue integrity,
activation of apoptotic pathways and pathologic consequences (Zhou et al 1999).
It is hypothesized that this cell loss in the presence of ROS stress can be
countered by an efficient cell adhesion. Thus, components of the matrix with an increased
cell adhesive ability could provide some form of protection to stress-exposed cells. The
increased cell survival, in turn can also have a substantial effect on cell movement during
in vivo stress-generating events like tissue remodelling and wound closure.
The physiological effects of oxidative stress in vitro can be mimicked by the use
of exogenously administered H2O2 and by heavy metals. ROS stress can be generated by
metals through two distinct mechanisms: With redox-active metals like Fe and Cr, a
141
Fenton-like reaction produces free radicals while redox-inactive toxic metals such as Hg
and Cd act through depletion of major antioxidant components leading to unquenched
free radicals (Ercal et al 2001). For this study, oxidative stress has been created via three
systems; subjecting cells to H2O2, a combination of Fe/ H2O2 and through heavy metals.
Two cryptic peptides isolated earlier; E1 (Chapter 3 and 5) and C2 (Chapter 5),
both displaying cell adhesion abilities was used as possible „stress-relieving‟ agents. The
present study was carried out to determine the cyto-protective and de-stressor activity of
the these peptides in countering stress generated by ROS along with the ability to affect
in vitro wound healing properties.
6.2 MATERIALS AND METHODS
T-flasks (Nunclon surface) were procured from Nunc, Roskilde, Denmark and
disposable culture dishes (35×10mm) were obtained from Fischer Scientific, Hanover
park, IL, USA. DMEM supplemented with 2mM glutamine and 10X antibiotic-
antimycotic solution were obtained from HiMedia, India. Foetal bovine serum and 10X
sterile filtered trypsin-EDTA solution was obtained from Sigma-Aldrich. Vero and HeLa
cell lines were procured from National Centre for Cell Science, Pune, India. All bench
work associated with cell lines, including peptide coating have been carried out inside a
class II bio-safety cabinet (Clean Air Systems, Chennai, India) for maintaining sanitized
conditions.
6.2.1 PEPTIDE ISOLATION AND COATING
The peptides E1 and C2 were isolated as described in Chapter 3 and 5
respectively. The purified peptides were coated onto the disposable dishes according to
standard procedures described in Chapter 5. A coating density of 0.507nmole cm-2
area,
known to display maximal cell adhesion has been used throughout this study.
6.2.2 CELL MAINTENANCE
Vero and HeLa cell lines were maintained as mentioned in section 5.2.1.2,
Chapter 5. For experiments, cells reaching 80% confluence were detached from T-flasks
with trypsin-EDTA, centrifuged and cell number enumerated by Neuber‟s chamber. The
viability was tested by trypan blue exclusion assay.
142
6.2.3 HYDROXYL RADICAL SCAVENGING ASSAY
The hydroxide radical scavenging assay was performed according to Zhang et al
(2010). A known quantity of peptides C2 and E1 were dissolved in 20mM KH2PO4-KOH
buffer, pH 7.5 in various dilutions ranging from 0.1-100mM. To 0.1ml of each solution,
the following reagents were added in order; 0.1ml of 1mM EDTA, 10mM H2O2, 75mM
2-deoxy-D-ribose, 2mM ascorbic acid and 1mM FeCl3. The reaction mixture was
incubated for 1h at 37°C and stopped by addition of 0.25ml of 20% TCA. For colour
development, 1ml of 1% TBA was added and the tubes with the reaction mixture were
placed in a boiling water bath for 15min. Absorbance was measured at 532nm after
cooling to room temperature. For blank, water was used instead of FeCl3 in a similar
reaction mixture. BSA and bovine tendon collagen were used as „negative control‟.
Collagen hydrolysate obtained after proteolysis was used as „test control‟ and BHT was
used as the „positive control‟. Assays were done in triplicates. Scavenging ability was
calculated from the following equation:
Radical scavenging activity (%) = 1001
Control
Test
Test is the absorbance of the test peptide samples and Control is the absorbance without
the peptides.
6.2.4 DE-STRESSOR ACTIVITY OF THE PEPTIDES
Stress was generated by five different stress-creating agents as listed in Table 6.1.
Concentrations of the stressors used were chosen from previously reported toxicity levels
(Levis and Majone 1979; Houot et al 2001; Hultberg et al 2001; Jungas et al 2002). Metal
stock solutions were prepared in deionized water and sterilized by filtration through
0.2µm filter. At the time of treatment, stock solutions were diluted in pre-warmed culture
medium to the final concentration required. For agents 4 and 5, stock H2O2 was stored in
4°C and diluted in 0.2M sterilized PBS before adding to the medium.
143
Table 6.1 List of agents used as stressors (*- concentration given in µM).
Agent Stressor Range of final concentrations (mM)
1 Cr (VI) 0 0.01 0.1 1 10 100
2 Fe (II) 0 0.01 0.1 1 10 100
3 Hg (II) 0 1.5625* 3.125
* 12.5
* 25
* 50
*
4 H2O2 0 2.5 5 10 25 100
5 Fenton‟s
reagent
Fe (II) 0 0.1 5 10 50 100
H2O2 0 0.01 0.1 1 10 50
Table 6.2 Experimental set-up for in vitro wound healing studies for three conditions;
in the absence of a stressor, in the presence of stressor and in the presence of both stressor
and de-stressor.
Set I
C2 coated E1 coated Collagen coated Uncoated
Set II
C2 coated +
H2O2
E1 coated +
H2O2
Collagen coated
+ H2O2
Uncoated +
H2O2
Set III
C2 coated +
E1 dissolved +
H2O2
E1 coated +
E1 dissolved +
H2O2
Collagen coated
+ E1 dissolved +
H2O2
Uncoated +
E1 dissolved
+ H2O2
144
6.2.4.1 ASSAYING DE-STRESSOR ACTIVITY IN COATED FORM
3.5×106
cells were seeded onto the coated dishes followed immediately by
exposing them to stressor agents 1-5 as per the final concentration given in Table 6.1.
The total volume was kept constant at 1.5ml. Cells seeded onto CCS dishes without
exposure to any stress was used as the control cell count. After 6h incubation, the adhered
cells were trypsinized and counted by haemocytometry.
6.2.4.2 ASSAYING DE-STRESSOR ACTIVITY IN DISSOLVED FORM
3.5×106
cells suspended in DMEM were seeded on CCS dishes and subjected to
the same level of stress as in section 6.2.4.1. Based upon activity of E1 in section 3.3.3.2,
Chapter 3, 0.0815µmoles (0.2mg ml-1
) of the active peptides E1 and C2 were solubilized
separately in 0.5ml medium and added to the cells. Care was taken to adjust the added
amount of stressors and cells such that the final concentration and volume to be the same
as before. After 6h incubation, adherent cells were counted and stressor concentration
ensuing 50% cell survival was calculated.
6.2.5 WOUND CLOSURE ASSAY
The wound closure assay was performed according to the protocols given by
Liang et al (2007). 3.5×105cells were seeded onto C2 and E1 coated dishes. An equal
number of cells were seeded onto collagen coated (positive control) and uncoated dishes
(negative controls). When cells reached 90-95% confluence, two parallel scratch wounds
of approximately 400µm diameter were made with a pipette tip in all the dishes. The
medium was replenished for all the dishes with certain changes as given in Table 6.2.
10mM H2O2 was added in set I, 10mM H2O2 along with E1 in dissolved form at a
concentration of 0.0815µmole/ml (based upon activity of E1 in section 6.2.4.2) was
added in set II and set III was incubated „as is‟. Images of a fixed area of the wound were
taken at regular intervals over the course of 24h. Image analysis was done by ImageJ
software (http://rsbweb.nih.gov/ij/) from the National Institute of Health, USA.
The wound-edge positions of the cells were averaged by digitally drawing lines. The area
of the wound was calculated as a rectangle, whose length was taken to be the length of
the wound in focus and breadth the average distance between the two advancing edges of
the wound. The decrease in this rectangular area with time was calculated by measuring
the decreasing distance between the advancing edges. Finally, the total was subtracted
145
from the original area to determine the area covered and expressed in % (Valster et al
2005).
6.2.6 STATISTICAL ANALYSIS
The assays were done in triplicates and activities reported as mean ± standard
deviation. Duplicate dishes were used for the wound healing migration assay. Larger
datasets were analyzed for statistical significance using one way and two-way ANOVA.
Comparison between two groups was accomplished by post-hoc Tukey‟s test and
student‟s t-test. P values less than 0.05 were considered significant.
6.3 RESULTS AND DISCUSSIONS
6.3.1 HYDROXYL RADICAL SCAVENGING ASSAY
E1 has already been confirmed in Chapter 3 as a moderately strong antioxidative
agent with radical scavenging, metal chelation and reductive ability. Since most ROS
stress generates •OH radicals, it was necessary to check for E1‟s ability to scavenge
•OH.
As displayed in Fig. 6.1, BHT, used as positive control, displayed highest activity
followed by E1 and the hydrolysate. The activities of BHT, the peptides and collagen
were significantly different (p<0.004) at a confidence level of 95% based on ANOVA. At
100nmole, E1 displayed a scavenging activity of 58.7%, whereas C2 achieved only 15%.
The activity of the hydrolysate was probably due to the numerous small constituent
charged peptides. However, it was lower than that of E1 (p<0.01) probably due to the
number of active sites being less when compared to that of E1. The results confirmed E1
to be a potent ROS stress releiver in the dissolved form with the ability to quench •OH.
146
0
10
20
30
40
50
60
70
80
90
BHT C2 E1 Hydro Coll
Samples
% s
caven
gin
g a
cti
vit
y 1 nmole
10 nmole
100 nmole
Fig. 6.1 Hydroxyl radical scavenging activity of C2, E1 and the hydrolysate (Hydro).
BHT and collagen (Coll) were used as positive and negative controls, respectively.
6.3.2 DE-STRESSOR ACTIVITIES OF PEPTIDES
Stress is a condition in which the homeostasis maintained in living systems fail,
giving rise to diverse pathological and physiological consequences. The efficacy of the
peptides in relieving oxidative stress generated through H2O2 and heavy metals has been
studied in this chapter. The photographs depicting the effect of the peptides in countering
stress are displayed in Fig. 6.2 (countering heavy metal-generated stress) and Fig. 6.3
(countering H2O2 and Fe (II)/ H2O2 - generated stress). Cell count in the CCS dishes were
found to decrease with increasing stressor concentration (correlation = -0.8). Presence of
the peptides; both in coated and in dissolved form had a positive impact on cell adhesion
and survival (correlation=-0.4 to -0.6) as displayed in Fig. 6.4-6.7.
147
25µM25µM25µM1.5µM
10mM10mM10mM0.1mM
10mM10mM10mM0.1mM
Hg (II)
Fe (II)
Cr (VI)
CCS + E1 in
dissolved form
C2 coatedCCSCCS
25µM25µM25µM1.5µM
10mM10mM10mM0.1mM
10mM10mM10mM0.1mM
Hg (II)
Fe (II)
Cr (VI)
CCS + E1 in
dissolved form
C2 coatedCCSCCS
Fig. 6.2 Photomicrographs depicting cell adhesion under heavy metal-generated stress.
The bar in the bottom right photomicrograph represents a length of 0.1mm.
1mM / 10mM1mM / 10mM1mM / 10mM0.01mM / 0.1mM
10mM10mM10mM2.5mM
HP +
Fe (II)
HP
CCS + E1 in
dissolved form
C2 coatedCCSCCS
1mM / 10mM1mM / 10mM1mM / 10mM0.01mM / 0.1mM
10mM10mM10mM2.5mM
HP +
Fe (II)
HP
CCS + E1 in
dissolved form
C2 coatedCCSCCS
Fig. 6.3 Photomicrographs depicting cell adhesion under HP (hydrogen peroxide) and Fe
(II) + HP generated stress. The magnification is the same as before.
148
a.
0
5
10
15
20
25
30
35
100 10 1 0.1 0.01 0
Cr(VI) mM
HeL
a c
ell c
ou
nt×
10
5
C2 ct C2 dv E1 ct E1 dv CCS
b.
0
5
10
15
20
25
30
35
100 10 1 0.1 0.01 0Fe (II) mM
HeL
a c
ell c
ou
nt×
10
5
c.
0
5
10
15
20
25
30
35
50 25 12.5 3.125 1.5625 0
Hg(II) µM
HeL
a C
ell c
ou
nt×
10
5
Fig. 6.4 Effect of increasing metal-generated stress on HeLa cells. Adhesion pattern of
cells in the presence of a. Cr (VI), b. Fe (II) and c. Hg (II). The term „ct‟ represents the
dishes with coated peptides while „dv‟ represents dishes with dissolved peptides.
149
a.
.
0
5
10
15
20
25
30
35
100 10 1 0.1 0.01 0
Cr(VI) mM
Vero
cell c
ou
nt×
10
5
C2 ct C2 dv E1 ct E1 dv CCS
b.
0
5
10
15
20
25
30
35
100 10 1 0.1 0.01 0
Fe(II) mM
Vero
cell c
ou
nt×
10
5
c.
0
5
10
15
20
25
30
35
50 25 12.5 3.125 1.5625 0
Hg(II) µM
Vero
cell c
ou
nt×
10
5
Fig. 6.5 Effect of increasing metal-generated stress on Vero cells. Adhesion pattern of
cells in the presence of a. Cr (VI), b. Fe (II) and c. Hg (II).
150
a.
0
5
10
15
20
25
30
35
100 25 10 5 2.5 0H2O2 mM
HeL
a c
ell c
ou
nt×
10
5
C2 ct C2 dv E1 ct E1 dv CCS
b.
0
5
10
15
20
25
30
35
100 25 10 5 2.5 0H2O2 mM
Vero
cell c
ou
nt×
10
5
Fig. 6.6 Effect of H2O2-generated stress on a. HeLa and b. Vero cells.
151
a.
05
101520253035
100 50 10 5 0.1 0
50 10 1 0.1 0.01 0
Fe(II) and H2O2 mM
HeL
a c
ell c
ou
nt×
10
5C2 ct C2 dv E1 ct E1 dv CCS
b.
05
101520253035
100 50 10 5 0.1 0
50 10 1 0.1 0.01 0
Fe(II) and H2O2 mM
Vero
cell c
ou
nt×
10
5
Fig. 6.7 Effect of Fe (II)/ H2O2 generated stress on a. HeLa and b. Vero cells.
Table 6.3 Stressor concentrations corresponding to 50% cell adhesion for HeLa and Vero