Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing

<div><p>PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on <i>in vitro</i> studies and its role in angiogenesis <i>in vivo</i> has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6–8 week old PAK1^−/− and PAK1^+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1^−/− mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1^−/− mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1^−/− but not PAK1^+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1^−/− mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1^−/− and PAK1^+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.</p></div

Unimpaired repair of cutaneous ear wounds in PAK1^−/− mice.

<p>A) Recovery of 2.0 mm ear punch wounds is represented by (Area _{day X/}Area _{day 0}) ×100. There was a notably larger but not statistically significant wound diameter in PAK1^−/− mice compared to PAK1^+/+ mice on day 7. B) The wound edge was stained with Mason's trichrome and shows a very similar wound closure between the two groups by day 28. n = 3 for both PAK1^+/+ and PAK1^−/− groups.</p

PAK1^−/− plantar perfusion and limb function following severe HLI in 16 week-old mice is slightly impaired compared to PAK1^+/+ mice, suggesting mild impairment in neovascularization in the absence of PAK1.

<p>A) I/NI foot perfusion ratio is similar between PAK1+/+ and PAK1−/− mice immediately following surgery, but starts to diverge by days 3 and 7 days (B–C). Worse use and appearance scores in PAK1^−/− mice on days 7 and 14 reflect the overall impaired limb function due to impaired neovascularization. D) Laser Doppler images obtained on day 0 pre- and post-surgery, and on day 21 showing similar perfusion between PAK1^−/− (N = 8) and PAK1^+/+ (N = 8) mice.</p

PAK1^−/− plantar perfusion and function in model of severe HLI in 6–8 week-old mice is similar to PAK1^+/+ mice.

<p>A) I/NI (Ischemic/Non-Ischemic) plantar perfusion ratio is comparable between PAK1^−/− and PAK1^+/+ mice as measured by laser Doppler imaging from day 0, immediately after HLI surgery, and throughout the 21 days after HLI surgery. B) Limb use score was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112239#s2" target="_blank">Materials and Methods</a> and is equivalent between groups, where a higher score is observed within the first 2 weeks after surgery and decreased significantly thereafter, and indicating recovery of limb function. C) Appearance scores of PAK1^+/+ and PAK1^−/− mice were not statistically significant. D) Laser Doppler images obtained immediately following HLI surgery and on day 21 reflect a lack of difference in perfusion between PAK1^+/+ (N = 8) and PAK^−/− (N = 11) mice.</p

Increased protein expression and phosphorylation of PAK2 in ischemic gastrocnemius muscle in PAK1^−/− compared to PAK1^+/+ mice.

<p>A) Western blots showing upregulated PAK2 expression and a trend towards increased phospho-PAK2 in PAK1^−/− mice (lanes represent samples from 3 different mice). We did not observe expression of PAK3 in gastrocnemius tissue; however, abundant PAK3 expression is found in mouse brain tissue as can be seen from a positive control sample in lane 7 of the PAK3 blot. B–C) Densitometry analysis reveals a 2-fold increase in PAK2 expression in PAK1^−/− compared to PAK1^+/+ mice, normalized to GAPDH as a loading control, and a concomitant increase in phospho-PAK2 relative to total PAK2. *indicates p≤0.05 using Student's T-test, n = 6. D) Densitometric analysis of total PAK2 protein expression in <i>non-ischemic</i> muscle did not reveal a change in PAK1^+/+ versus PAK1^−/− mice.</p

Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing - Figure 4

<p>A) A notable activation of ERK1/2 but not AKT is observed in PAK1−/− gastrocnemius tissue one hour after HLI surgery. Western blotting analysis of PAK1^−/− non-ischemic and ischemic gastrocnemius muscle shows enhanced phosphorylation of ERK1/2 on Thr202/Tyr204 compared to PAK1^+/+ tissue, whereas phosphorylation of AKT was unchanged. Lanes are grouped into Non-Ischemic and Ischemic from either PAK1^+/+ and PAK1^−/− mice. B and D) Densitometric analysis of non-ischemic tissue from PAK1^+/+ and PAK1^−/− show an increase in pERK1/2 activation (p = 0.03 and 0.06 for pERK1 and pERK2 respectively, Student's T-test) whereas a difference in pAKT activation is not observed. C and E) In ischemic tissue a trend towards an increase in pERK1 (p = 0.08, Student's T-test) but not pERK2 or pAKT in PAK1^−/− compared to control PAK1^+/+ was observed. A total of 3 PAK1^+/+ and PAK1^−/− mice were analyzed. Blots for pERK1/2 and pAKT were normalized to tERK1/2 and tAKT respectively.</p

Marked reduction in aortic EC sprouting following IPA3 treatment.

<p>A) Similar EC sprouting is observed in PAK1^−/− and PAK^+/+ aortic rings cultured for 5 days in growth media supplemented with FBS, whereas a notable reduction in sprouting was observed following 30 µM IPA3 treatment. B) Magnification of the selected region showing EC sprouts (white arrows). C) Quantification of EC sprouts shows less EC sprouts in PAK1^+/+ and PAK1^−/− with IPA3 treatment. Data points represent the average values ±SEM from 3–9 rings from 3–4 mice per group; * denotes p<0.05 compared to FBS alone.</p

Comparison of Lung Histology for WT and <i>Pak1</i>^−/− mice.

<p>Airway wall area (A), airway smooth muscle area (B), and airway epithelium area (C) were not significantly different between the WT (black squares) and <i>Pak1</i>^−/− (grey circles) mice when assessed by ANOVA adjusting for airway perimeter (p>0.15). (N = 5 mice in each group).</p

Aerosolized IPA3 inhibited airway contractility <i>in vivo</i> and suppressed Pak activation.

<p>When assessed by repeated ANOVA, resistance increased with increasing ACh dose (p<0.0001), and IPA3 dissolved in 1% DMSO (N = 3) and aerosolized 1-hour prior to bronchial challenge of WT mice significantly reduced the slope of the increase in resistance (p<0.0001), as well as the magnitude of the increase in resistance compared to control vehicle (1%DMSO; N = 5) (p<0.001) (A). Post-hoc analysis indicated that IPA3 treatment resulted in lower resistances at MCh doses ≥33 mg/ml (p<0.05). Tracheal smooth muscle isolated from WT mice treated <i>in vivo</i> with IPA3 demonstrated significantly lower Pak activation as assessed by Pak T423 phosphorylation following stimulation with ACh compared to airway smooth muscle isolated from WT mice (B). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042601#s2" target="_blank">Results</a> represent 2 samples of tracheal smooth muscle from each group. Each sample consisted of pooled tracheal muscle tissues from 3 separate mice with the same treatment.</p

Tracheas isolated from <i>Pak1</i>^−/− mice showed reduced contractility to ACh in vitro.

<p>Isometric force generation (% of maximal force to ACh stimulation in WT mice) was significantly lower in tracheas isolated from <i>Pak1</i>^−/− (grey squares) mice compared to WT (black diamonds) mice (N = 10 or 11 in each group, p<0.01).</p