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Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

IntroductionAngiogenesis is involved in physiologic processes such as embryogenesis, wound healing, and the female reproductive cycle, and also contributes to the pathogenesis of numerous disorders, including atherosclerosis, ischemic disease, arthritis, and cancer. 1 Normally, angiogenesis is strictly controlled by the balance between angiogenic promoters and inhibitors.Thrombomodulin (TM) is a type I-glycosylated membrane protein composed of 5 distinct domains. At the NH 2 -terminal is a C-type lectin-like domain followed by 6 consecutively repeated epidermal growth factor (EGF)-like domains and an O-glycosylation site-rich domain. Connected to these extracellular domains is a transmembrane domain followed by a short cytoplasmic tail. 2 TM functions as part of the anticoagulant pathway; it binds thrombin on the cell surface and catalyzes proteolytic activation of protein C. 2 Soluble TM fragments have been detected in the medium of cultured TM-expressing cells 3 and human plasma and urine, 4-6 but the physiologic significance of the soluble TM fragments remains unclear. Our previous study showed that the recombinant EGF-like domain plus the O-glycosylation site-rich domain of TM (rTMD23) promotes angiogenesis in vitro and in vivo. 7 The multiple mechanisms underlying the anti-inflammatory activity of the recombinant lectin-like domain of TM have been demonstrated recently. [8][9][10][11] Lewis Y Ag (LeY) belongs to the type II Lewis Ag family, which is structurally related to the determinants of the ABH blood group system. LeY is expressed on the cell membrane along with phospholipids, 12 EGF receptors, 13 and mucins, 14 and its soluble form is up-regulated in the synovial fluid of human angiogenic rheumatoid arthritis. 15 LeY has been suggested to be a participant in cell-cell interaction, 16 arthritis, 15 and cancer. 17 In addition, ablation in endothelial cells of fucosyltransferase I, which is crucial for generation of the precursor of type II Lewis Ag, resulted in defective tube formation, suggesting the role of LeY in endothelial cell-cell contacts. 18 Previously, we demonstrated that recombinant lectin-like domain of TM (rTMD1) suppressed lipopolysaccharide-induced inflammation through interaction with LeY-conjugated lipopolysaccharide. 11 However, the biologic significance of the interaction between rTMD1 and LeY on the endothelial cell surface has never been investigated. In this study, the biologic function of LeY on endothelial cells and the antiangiogenic function of rTMD1 were investigated. In addition, recombinant adeno-associated virus (AAV) expression vector carrying TMD1 (AAV-TMD1) resulted in efficient suppression of angiogenesis with a single injection of AAV in mice. An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance ...

Toll-like receptor (TLR) family plays a key role in innate immunity and various inflammatory responses. TLR4, one of the well-characterized pattern-recognition receptors, can be activated by endogenous damage-associated molecular pattern molecules such as high mobility group box 1 (HMGB1) to sustain sterile inflammation. Evidence suggested that blockade of TLR4 signaling may confer protection against abdominal aortic aneurysm (AAA). Herein we aimed to obtain further insight into the mechanism by which TLR4 might promote aneurysm formation. Characterization of the CaCl2-induced AAA model in mice revealed that upregulation of TLR4 expression, localized predominantly to vascular smooth muscle cells (VSMCs), was followed by a late decline during a 28-day period of AAA development. In vitro, TLR4 expression was increased in VSMCs treated with HMGB1. Knockdown of TLR4 by siRNA attenuated HMGB1-enhanced production of proinflammatory cytokines, specifically interleukin-6 and monocyte chemoattractant protein-1 (MCP-1), and matrix-degrading matrix metalloproteinase (MMP)-2 from VSMCs. In vivo, two different strains of TLR4-deficient (C57BL/10ScNJ and C3H/HeJ) mice were resistant to CaCl2-induced AAA formation compared to their respective controls (C57BL/10ScSnJ and C3H/HeN). Knockout of TLR4 reduced interleukin-6 and MCP-1 levels and HMGB1 expression, attenuated macrophage accumulation, and eventually suppressed MMP production, elastin destruction and VSMC loss. Finally, human AAA exhibited higher TLR4 expression that was localized to VSMCs. These data suggest that TLR4 signaling contributes to AAA formation by promoting a proinflammatory status of VSMCs and by inducing proteinase release from VSMCs during aneurysm initiation and development.