Effects of human pericytes in a murine excision model of wound healing (2015)

We previously reported that dermal pericytes profoundly enhance the regenerative capacity of human keratinocytes, above that of dermal fibroblasts in an angiogenesis-free organotypic model 1, a property quite distinct from their well-known role in regulating blood vessel structural stability. Given the mesenchymal stem cell/MSC-like multilineage differentiation capacity of pericytes 1, 2, we hypothesised that pericytes represent a potent stem cell population with a pro-proliferative role in organ growth, repair and regeneration. Do dermal pericytes promote healing in an excision wound model? Four mm punch ‘biopsies’ of human neonatal foreskin dermal CD45−VLA-1/α1/CD49abri pericytes, CD45−VLA-1dim fibroblasts or both cell types 1, implanted into collagen type 1 gels, were placed directly onto 4 mm excisional wounds on the dorsal skin of BALB/c SCID male mice and secured in place with gauze and 3M Tegaderm™. Wounds were left to heal and harvested at 3 and 7 days (d3/7) postwounding. Wound healing assessed by measuring wound widths at d3 (Fig. 1a,c) or wound areas and rate of re-epithelialization showed no statistically significant differences when inoculated with fibroblasts, pericytes or a combination of both, compared to collagen controls (n = 3 independent experiments). By d7, although all wounds showed an overall decrease in wound width compared to d3, and complete re-epithelialisation, the pericyte-inoculated wounds displayed the greatest wound width of all conditions (Fig. 1b,d), suggesting a delay in dermal wound resolution in their presence. Notably, Ki67+ epidermal cells were abundant in all the wounds, with no significant differences in their numbers in the presence of pericytes (data not shown). Importantly, we confirmed that the inoculated human fibroblasts and pericytes were still present in the collagen gels at d7 (not detected in collagen controls), albeit at low frequency, using in situ hybridization for the human ALU element, (Fig. 2a). Given the importance of decreased inflammation in the later stages of wound healing, we stained and quantified the number of murine neutrophils (Ly-6G) and macrophages (Mac-3) in the wounds. Notably, pericyte-inoculated wounds showed an increase in neutrophil numbers as early as d3 (P = 0.04), as did fibroblast-treated wounds (P = 0.014) compared to collagen controls, but not in the combined presence of pericytes and fibroblasts. Interestingly, by d7, pericyte-treated wounds continued to display a trend towards higher numbers of neutrophils compared to pericyte + fibroblast (Fig. 2b) not achieving statistical significance (P = 0.08). Concomitantly, macrophage numbers remained elevated in the pericyte-treated wounds compared to fibroblast (P = 0.002) and combined pericyte-/fibroblast-treated wounds (P = 0.0013) at d7 (Fig. 2c). One possibility is that pericytes induce a prolonged inflammatory response – we sought to confirm this by determining whether the expression of a central regulator of wound healing, that is TGF-β1 and Smad3 – one of its major downstream targets affecting key tissue remodelling processes 3. Although the TGF-β family of growth factors has a complex role in regulating specific stages of wound healing, persistent Smad3 expression can delay wound healing by affecting monocyte infiltration. Specifically, Smad3-deficient mice show improved wound healing with an increased rate of re-epithelialisation and reduced monocyte infiltration 4. Immunostaining revealed a significant increase in TGF-β1 levels in the pericyte-treated wounds with higher numbers of TGF-β1+ cells compared to fibroblast (P < 0.01) and combined pericyte + fibroblast-treated wounds (P < 0.001) at d7 (Fig. 2d). Similarly, greater numbers of Smad3 expressing cells were present in pericyte-treated wounds, with statistically significant differences between the pericyte alone and combined pericyte + fibroblast-treated wounds (P < 0.01; Fig. 2e,f). Our data show that pericyte treatment delays the rate of dermal wound resolution (while re-epithelialisation and wound closure remain unaffected), possibly through a failure to downregulate the inflammatory response, although further experimentation is needed to resolve the specific nature of the immune response following human pericyte introduction in a murine background. Interestingly, while TGF-β1 null mice do not exhibit impaired wound healing, they exhibit signs of decreased inflammation, that is reduced granulation tissue correlated with increased re-epithelialisation rates, suggesting that endogenous TGF-β1 may increase inflammation and retard wound closure 5. Thus, we did not find any beneficial function for pericytes in cutaneous wound healing. While it seems counter-intuitive that the constitutively α-SMA+ (α-smooth muscle actin) pericyte population should serve a contractile function resulting in more rapid wound healing, the distribution of human pericytes as single cells in the wounds not localised perivascularly, and perhaps in insufficient numbers to act as myofibroblasts as reported 6, supports our observation that wound closure was not significantly accelerated in the presence of pericytes. Interestingly, our data are also consistent with other work concluding that endogenous pericytes do not aid wound contraction as pericytes migrating into wounds away from blood vessels, lacked α-SMA, promoting fibrosis through collagen production 7. Indeed, a recent review strongly implicates pericytes from various organs in fibrosis following injury 8, including evidence for the activation of a pro-inflammatory PDGFRα+/ADAM12+ subset of perivascular cells in acute skin and muscle injury 9. Although our previous work showed that pericytes promote epithelial cell proliferation 1, the rate of re-epithelialisation and the number of Ki67+ epithelial cells was unaltered in this wound healing model-it is possible however, that factors secreted by exogenous human pericytes cannot efficiently signal to the host murine keratinocytes to influence epithelial regeneration. Notably, our previous study utilised an in vitro organotypic culture model free from systemic factors including immune cells and resulting pro-inflammatory cytokines. Interestingly, the introduction of fibroblasts either alone or in combination with pericytes – perhaps representing a more physiological mesenchymal microenvironment – showed a better dampening down of infiltrating macrophages correlated with decreased wound width at later stages of healing, notwithstanding the potential contribution of other mesenchymal populations (reviewed in Ref. 10). Thus, while the use of pericytes to enhance ex vivo epidermal regenerative capacity for autologous transplantation remains a possibility as suggested by our previous work 1, this pilot study indicates that an imbalance in the mesenchymal microenvironment can prolong dermal wound resolution. SJM and PK designed the study, SJM, LZ, PA, PK and AJC performed the research, SJM, LZ, PA, PK and AJC performed data analysis, and PK, SJM and AJC wrote and revised the study. The authors state no conflict of interest.