Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor–like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis

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IntroductionPlatelet activation is thought to be a key event in acute vascular thrombosis. Therefore, prevention of enhanced platelet activation is a major target of therapeutic strategies fighting cardiovascular and cerebrovascular diseases. [1][2][3] An important stimulus for physiologic platelet activation and thrombus formation is the contact of platelets with components of the subendothelial matrix, like collagen. 4 Although Marcus et al have shown as early as 1977 that platelets have the ability to release superoxide anions (O 2 Ϫ ), 5 it was only recently proposed that platelets stimulated by collagen produce reactive oxygen species (ROS) such as hydrogen peroxide, 6 hydroxyl radicals, 7 or O 2 Ϫ . 7,8 While O 2 Ϫ , a highly reactive radical, damages cells in high concentrations by reacting with proteins, lipids, and DNA, in low concentrations its continuous production, with similarity to second messengers, has been suggested to indirectly affect signal transduction processes. 9,10 Platelet agonists other than collagen, such as thrombin or ADP, do not seem to induce ROS formation during aggregation. 8 This difference raises the question whether O 2 Ϫ formation could serve a modulating function when thrombus formation is induced by collagen.The cellular source of platelet O 2 Ϫ is unclear. Growing evidence supports the assumption that platelet activation by collagen is specifically due to binding to the glycoprotein VI (GPVI)-receptor, 11,12 resulting in a cascade of tyrosine phosphorylation events ultimately leading to activation of phospholipase C␥ (PLC␥), 13 which is known to strongly activate protein kinase C (PKC) through production of diacylglycerol. 4 Recently, evidence for the existence of a neutrophil-type reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase in platelets that can be activated by PKC and is involved in O 2 Ϫ formation has been presented, [14][15][16] similar as in other O 2 Ϫ -generating systems, like the vascular endothelium. In endothelial cells, an NAD(P)H oxidase is the main source of O 2 Ϫ . 17 As O 2 Ϫ readily reacts with NO, this has been suggested to result in attenuated 18,19 and the role of O 2 Ϫ in the regulation of vascular tone has become a major focus of interest. 17 Moreover, antioxidants like N-acetylcysteine (NAC) have been shown to exert direct antiaggregatory effects. 20 Although these findings raise the possibility that platelet-derived O 2 Ϫ is involved in regulating platelet activation, evidence for a role of platelet-derived O 2 Ϫ in platelet function is rare. In a canine model of coronary arterial thrombosis, thrombus formation was regulated by intraplatelet redox state. 21 Leo and colleagues have shown that platelets subjected to anoxia/ reoxygenation are more reactive, due to an enhanced O 2 Ϫ generation. 14 However, so far it remains unclear whether an enhanced O 2 Ϫ production occurs also during direct platelet activation, such as with collagen, and how this could affect thrombus growth. Whereas Supported by a grant from the Friedrich-...

Delivery of antisense oligodesoxynucleotides (ODN) into primary cells is a specific strategy for research with therapeutic perspectives but transfection-associated difficulties. We established the technique of magnetofection to enhance ODN delivery at low toxicity and procedure time in vitro and in vivo. In vitro, target knockout was assessed at protein and mRNA levels and by measuring superoxide generation after antisense magnetofection against the p22(phox) subunit of endothelial NAD(P)H-oxidase. Under magnetic field guidance, low-dose magnetic particle-bound ODN were transfected to 84% human umbilical vein endothelial cells within 15 min followed by nuclear accumulation within 2 h, which required 24 h using standard methods. Antisense magnetofection against p22(phox) significantly decreased basal and prevented stimulated superoxide release due to loss of NAD(P)H-oxidase activity by mRNA knockout as assessed after 24 h. Knockout of endothelial phosphatase SHP-1 and connexin 37 proteins confirmed the method's efficiency. Transfection-associated toxicity was minimal. Twenty-four hours after injection of fluorescence-labeled ODN into femoral arteries of male mice, there was specific ODN uptake only into cremaster vessels exposed to magnetic fields during injection. Magnetofection is an ideal tool for delivery of functionally active ODN to difficult-to-transfect cells to study gene/protein function and a promising strategy for targeted ODN delivery in vivo.