Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.

PECAM1(+)/Sca1(+)/CD38(+) vascular cells transform into myofibroblast-like cells in skin wound repair.

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1(+)/Sca1(+) vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1(+)/Sca1(+)/CD38(+) cells proliferate upon wounding and could give rise to α-SMA(+) myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1(+)/Sca1(+)/CD38(+) vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1(+)/Sca1(+)/CD38(+) cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.

Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif.

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.

Differential proteomic analysis distinguishes tissue repair biomarker signatures in wound exudates obtained from normal healing and chronic wounds.

Chronic wounds associated with vascular disease, diabetes mellitus, or aging are leading causes of morbidity in western countries and represent an unresolved clinical problem. The development of innovative strategies to promote tissue repair is therefore an important task that requires a more thorough analysis of the underlying molecular pathophysiology. We propose that the understanding of the complex biological events that control tissue repair or its failure largely benefits from a broad analytical approach as provided by novel proteomic methodologies. Here we present the first comparative proteome analysis of wound exudates obtained from normal healing or nonhealing (venous leg ulcer) human skin wounds. A total of 149 proteins were identified with high confidence. A minority of proteins was exclusively present in exudate of the healing wound (23 proteins) or the nonhealing wound (26 proteins). Of particular interest was the differential distribution of specific proteins among the two different healing phenotypes. Whereas in the exudate obtained from the healing wound mediators characteristic for tissue formation were abundantly present, in the exudate obtained from the nonhealing wound numerous mediators characteristic for a persistent inflammatory and tissue destructive response were identified. Furthermore, the study also revealed interesting results regarding the identification of new proteins with yet unknown functions in skin repair. This analysis therefore represents an important basis for the search for potential biomarkers, which give rise to a better understanding and monitoring of disease progression in chronic wounds.