Human Platelet-Rich Plasma- and Extracellular Matrix-Derived Peptides Promote Impaired Cutaneous Wound Healing In Vivo

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.National Institutes of Health (U.S.) (Grant EY15125)National Institutes of Health (U.S.) (Grant EY19533)Wound Care Partners, LL

Expression, Turn-Over, Localization, and Transport of Pocilloporins in Reef Building Corals

Coral reefs are a critical resource to developing and developed nations world wide. Providing shelter, food, monetary value, and a vast resource of ecological wealth, the corals of the reefs underpin an entire ecosystem. Climate change, driven by increased greenhouse gases, is raising the temperature of Earth’s waters and atmosphere, while making the planet’s oceans increasingly acidic. Brightly lit and increasingly warm tropical waters present a potentially challenging environment in which scleractinian corals grow. In attempting to cope with the competing stresses of intense photon flux density (PFD) and anomalously high sea surface temperatures, corals and dinoflagellates exhibit myriad biochemical and physiological adaptations. Pocilloporins, a diverse group of non-fluorescent green fluorescent protein (GFP) homologs found across Cnidaria and beyond, are one such adaptation within the tissues of heavily pigmented scleractinian corals. Chemically unique amongst pigments, GFP-like pigments exist as pure protein chromophores and exhibit little to no cytotoxicity when naturally occurring. This non-fluorescent class of GFP-like pigments has found popularity in biochemical and biotechnological applications, though an ecological and evolutionary explanation for the heavy conservation of pocilloporins across a broad range of scleractinian corals and related cnidaria is still a subject of scientific research and debate. This thesis supports the hypothesis that pocilloporins act as a naturally occurring photoprotective pigment in reef-building corals, specifically acting to filter and regulate the light environment within coral polyps. In examining the role of pocilloporins in Scleractinia, the need to examine environmental sources of pigment production induction and suppression, the localization of pigments within coral tissues and cells, and the ability of coral colonies to direct resource allocation with regards to pocilloporin production were identified as lines of inquiry. Briefly, for experiments examining either pocilloporin induction or suppression, the following aspects were studied: holobiont responses in the form of mRNA signal expression, host pigment isolation and analysis, dinoflagellate density and pigmentation sampling, and chlorophyll fluorescence of live corals. Blue morph Acropora aspera, common to the reef flat of Heron Island (Great Barrier Reef, Australia), were subjected to 99% shade and thermal bleaching threshold temperatures in separate attempts to suppress pocilloporin expression, while red morph Montipora monasteriata was transplanted at equivalent depth from their natural cave environments to exposed portions of the spur and groove formations of the northern face of Wistari Reef (Great Barrier Reef, Australia). Both ambient temperature and heat-stressed A. aspera were concurrently collected during the thermal stress experiment and placed in preservatives for immuno-histochemical localization of pocilloporins with their tissues. Finally, radio-labelled glycine, a very common amino acid in the primary sequence of pocilloporin, was injected into artificially injured tan morph Montipora monasteriata, also on the northern face of Wistrai Reef to study the uptake of dissolved organic materials (DOM) and incorporation of metabolic resources into newly generated pigments. Pocilloporins proved easier to induce in this work than suppress, and the location of these pigments in A. aspera tissues suggests a potential mechanism. The data demonstrated the presence of pocilloporins in the most directly exposed epidermal and gastrodermal tissues of the coral polyp, specifically the outermost layers of epidermis and gastrodermal layers bordering directly upon the gastrovascular cavity. Closer inspection through anti-pocilloporin-gold stained TEM images was highly suggestive of pocilloporin secretion in coral mucus, a theory separately supported by observations of coral mucus in collected live corals. Neither suppression experiment induced heavy mucus sloughing in A. aspera, so despite multi-fold reductions in pocilloporin mRNA as a result of applied stimuli, the continued presence of pocilloporin aaCP592 in blue morph A. aspera is not surprising. Conversely, pocilloporin msCP576 in plating Montipora monasteriata was induced in response to both general increases in PFD and specific increases of PFD at the sites of physical injury. Additionally, tan morph Montipora monasteriata demonstrated the capacity to collect and allocate DOM from the environment to assist in the production of new pigments and tissues, an energetically expensive process. The reduction of the orange-red spectrum in favour of the blue light ranges is generally beneficial to the photosynthetic systems of both higher plants and the resident dinoflagellates of corals. msCP576 and aa592, both positively identified as pocilloporins within this work, absorb within the orange-red region and apparently act as a photoprotective filter in all exposed surfaces of heavily pigmented corals, enhancing the blue spectrum of incident and reflected PFD and generally regulating the internal light environment

ECM-and platelet extract-derived peptides stimulate in vitro angiogenesis and epithelial proliferation.

<p>A – In vitro angiogenesis assay. Bovine capillary endothelial cells were plated on the surface of growth factor reduced Matrigel in the presence or absence of 100 nM comb1 or 250 nM UN3, or the two peptides combined. DMEM supplemented with 1% BCS was used as control, cells that have received DMEM/1% supplemented with 10 ng/ml bFGF or VEGF served as positive control. Total tube length was measured at 7 h post-plating. Relative tube length compared to control is shown. B – In vitro epithelial proliferation assay. Hacat cells were plated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032146#s2" target="_blank">Materials and Methods</a>. The peptides were added at 100 or 250 nM (comb1 and UN3 respectively). Cell counting was performed at 5 days post-plating. Relative cells numbers as compared to control are shown. Data are presented as mean +/− standard error; * - indicates p<0.05.</p

Histological evaluation of wound healing effects of the peptides in CY-treated Balb/c mice.

<p>Animals were either wounded and treated with CMC (A) or injected with CY 1 and 4 days prior to injury, wounded and treated with CMC alone (B), with Regranex (C), with UN3 (D), comb1 (E) or a combination of UN3 and comb1 (F). Wounds were excised at day 10 post-injury, formalin-fixed, sectioned and stained with haematoxylin and eosin. Dotted line delineates the wound bed. Scale bar 500 µm.</p

Characterization of Lot 1-derived fractions.

<p>A – silver stained SDS-PAGE demonstrating reduction of composition complexity following gel filtration and ion exchange chromatography. 1 - MW markers, 2 – unfractionated lot 1, 3- fraction 22, 4 – proteins eluted with 0.5 M NaCl. B – fractionated platelet extracts retain their biological activity toward epithelial cells. For proliferation assay NeoNHEK were plated in keratinocyte growth medium the presence or absence of unfractionated proteins from Lot 1 or fractions of Lot 1 (0.1–200 ng/mL) as indicated. HB-EGH (10 ng/mL) was used as positive control. Relative proliferation compared to control is shown. Data are presented as mean +/− standard error.</p

Characterization of protein composition of human platelet extracts.

<p>A – Coomassie Blue stained SDS-PAGE demonstrating the complexity of protein composition of platelet lysate and extracts. B – Western blot analysis of platelet lysate and extracts revealing the presence of myosin in lots 2 and 3, but not lot 1 extract or platelet lysate. 1- platelet lysate; 2 – platelet extract lot 1, 15 µg/mL; 3 - platelet extract lot 1, 50 µg/mL; 4 - platelet extract lot 2, 15 µg/mL; 5 - platelet extract lot 2, 50 µg/mL; 6 - platelet extract lot 3, 15 µg/mL; 7 - platelet extract lot 3, 50 µg/mL.</p

Platelet extracts stimulate epithelial proliferation in vitro.

<p>Hacat cells were plated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032146#s2" target="_blank">Materials and Methods</a> in the presence or absence of at 1 µg/mL of proteins from platelet extracts (Lots 1–3). Relative proliferation compared to control is shown. Data presented as mean +/− standard error; * - indicates p<0.05.</p

Quantitative evaluation of wound healing angiogenesis induced by the peptides.

<p>Immunohistochemistry for CD31 and HSPG was used to identify blood vessels in 10 days old wounds. The areas occupied by the blood vessels were identified by colocalized CD31 and HSPG staining and quantified using ImageJ. At least two wounds were quantified for each condition. Data are presented as mean +/− standard error; * - indicates p<0.05 compared to CY alone.</p