Sequential induction of pro- and anti-inflammatory prostaglandins and peroxisome proliferators-activated receptor-gamma during normal wound healing: a time course study

Abstract

Lipid mediators generated from metabolism of arachidonic acid play a crucial role in the initiating and resolution of acute inflammation by shifting from pro-inflammatory prostaglandin (PG) E2 to anti-inflammatory PGD2 and its metabolites. The changes in PG levels over time during the normal wound-repair process have not, however, been reported. We determined the temporal expression of PG and their biosynthetic enzymes using the full thickness incisional model of normal wound healing in mice. We demonstrate that during normal wound repair, there is a shift in the metabolism of arachidonate from PGE2 during the acute inflammatory phase to PGD2 during the repair phase. This shift is mediated by temporal changes in the expression of cyclooxygenases (COX) and microsomal PGES (mPGES)-1. Inducible COX (COX-2) expression is sustained throughout the initiation and repair process, but mPGES-1 is increased only during the acute inflammatory phase and its disappearance coincides with increased PGD2. PGD2 and its degradation products are known to mediate their anti-inflammatory effects by binding to peroxisome proliferators-activated receptor gamma (PPARgamma). In this study, we show that PPARgamma is upregulated during the resolution phase of wound repair concomitant with the shift to PGD2, and may be responsible for initiating endogenous mechanism resulting in healing/resolution.

Figure 1. Temporal protein expression profile of COX-2 and mPGES-1 during the time course of normal wound healing

Protein expressions of COX-2 and mPGES-1 in the isolated wound tissues were determined by western blotting during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). COX-2 and mPGES-1 proteins were normalized by β-actin which was used as an internal control to assess equal protein loading. * indicates statistical significance at P<0.05 at each respective time point compared to unstimulated control skin on day 0. Data are expressed as the mean ± SEM for n=4 separate blots.

Figure 2. Temporal mRNA expression profile of COX-2 and mPGES-1 during the time course of normal wound healing

mRNA expressions of COX-2 and mPGES-1 in the isolated wound tissues were determined by RT-PCR during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). COX-2 (25 cycles) and mPGES-1 (30 cycles) mRNA levels were normalized by GAPDH (20 cycles) which was used as an internal control. * indicates statistical significance at P<0.05 at each respective time point compared to unstimulated control skin on day 0. Data are expressed as the mean ± SEM for n=4 separate gels.

Figure 3. Localization of COX-2 and mPGES-1 in the day 3 wounds

COX-2 and mPGES-1 localization in the day 3 wound tissue was detected by immunohistochemistry. Fig 3a shows extensive staining of COX-2 in the dermal region of the wounded skin in the day 3 wound tissues. Fig 3b shows extensive staining of mPGES-1 in the epidermal region of the wounded skin in the day 3 wound tissues. WS= wound site, D=dermis, E=epidermis. → signifies the region of extensive staining. Magnification x10.

Figure 4. Temporal protein (a) and mRNA (b) expression profiles of COX-1, mPGES-2, cPGES, H-PGDS and PGIS during the time course of normal wound healing

(a) Protein and (b) mRNA expressions of COX-1, mPGES-2, cPGES, H-PGDS and PGIS were determined by western blotting and RT-PCR respectively during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). Proteins were normalized by β-actin (a) and mRNA expressions were normalized with GAPDH (b). Representative data for n=3 separate blots and gels is shown.

Figure 4. Temporal protein (a) and mRNA (b) expression profiles of COX-1, mPGES-2, cPGES, H-PGDS and PGIS during the time course of normal wound healing

(a) Protein and (b) mRNA expressions of COX-1, mPGES-2, cPGES, H-PGDS and PGIS were determined by western blotting and RT-PCR respectively during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). Proteins were normalized by β-actin (a) and mRNA expressions were normalized with GAPDH (b). Representative data for n=3 separate blots and gels is shown.

Figure 5. Temporal production profile of PGE₂ and PGD₂ during the time course of normal wound healing

The levels of PGE₂ (light bars) and PGD₂ (dark bars) in the wound tissues were determined by EIAs during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). Levels of PGs were normalized to mg of protein for each respective wound tissue. * indicates statistical significance at P<0.05 at each respective time point compared to unstimulated control skin on day 0. Data are expressed as the mean ± SEM for n=7 separate determinations.

Figure 6. Temporal expression of PPARγ during the time course of normal wound healing

(a) Protein and (b) mRNA expressions of PPARγ were determined by western blotting and RT-PCR respectively during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). PPARγ protein (a) and mRNA (b) expressions at each respective time points were normalized by β-actin and GAPDH respectively. * indicates statistical significance at P<0.05 at each respective time point compared to unstimulated control skin on day 0. Data are expressed as the mean ± SEM for n=4 separate blots. (c) PPARγ localization in the day 3 wound tissues was detected by immunohistochemistry. Extensive staining of PPARγ was observed in the epidermal region of the wounded skin in the day 14 wound tissues. WS= wound site, D=dermis, E=epidermis. → signifies the region of extensive staining. Magnification x10.

Figure 6. Temporal expression of PPARγ during the time course of normal wound healing

(a) Protein and (b) mRNA expressions of PPARγ were determined by western blotting and RT-PCR respectively during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). PPARγ protein (a) and mRNA (b) expressions at each respective time points were normalized by β-actin and GAPDH respectively. * indicates statistical significance at P<0.05 at each respective time point compared to unstimulated control skin on day 0. Data are expressed as the mean ± SEM for n=4 separate blots. (c) PPARγ localization in the day 3 wound tissues was detected by immunohistochemistry. Extensive staining of PPARγ was observed in the epidermal region of the wounded skin in the day 14 wound tissues. WS= wound site, D=dermis, E=epidermis. → signifies the region of extensive staining. Magnification x10.

Figure 6. Temporal expression of PPARγ during the time course of normal wound healing

(a) Protein and (b) mRNA expressions of PPARγ were determined by western blotting and RT-PCR respectively during a complete time course of 21 days comprising of: day 0 (Control unwounded skin), days 1 and 3 (inflammatory phase) and days 7, 14 and 21 (resolution phase). PPARγ protein (a) and mRNA (b) expressions at each respective time points were normalized by β-actin and GAPDH respectively. * indicates statistical significance at P<0.05 at each respective time point compared to unstimulated control skin on day 0. Data are expressed as the mean ± SEM for n=4 separate blots. (c) PPARγ localization in the day 3 wound tissues was detected by immunohistochemistry. Extensive staining of PPARγ was observed in the epidermal region of the wounded skin in the day 14 wound tissues. WS= wound site, D=dermis, E=epidermis. → signifies the region of extensive staining. Magnification x10.